RNA sequence-specific mediators of RNA interference

ABSTRACT

The present invention relates to a  Drosophila  in vitro system which was used to demonstrate that dsRNA is processed to RNA segments 21-23 nucleotides (nt) in length. Furthermore, when these 21-23 nt fragments are purified and added back to  Drosophila  extracts, they mediate RNA interference in the absence of long dsRNA. Thus, these 21-23 nt fragments are the sequence-specific mediators of RNA degradation. A molecular signal, which may be their specific length, must be present in these 21-23 nt fragments to recruit cellular factors involved in RNAi. This present invention encompasses these 21-23 nt fragments and their use for specifically inactivating gene function. The use of these fragments (or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for degradation in mammalian cells, where the use of long dsRNAs to elicit RNAi is usually not practical, presumably because of the deleterious effects of the interferon response. This specific targeting of a particular gene function is useful in functional genomic and therapeutic applications.

This application is a continuation of U.S. application Ser. No.11/474,930, entitled “RNA SEQUENCE-SPECIFIC MEDIATORS OF RNAINTERFERENCE” filed on Jun. 26, 2006 now abandoned, which is hereinincorporated by reference in its entirety. Application Ser. No.11/474,930 is a divisional of U.S. application Ser. No. 09/821,832,entitled “RNA SEQUENCE-SPECIFIC MEDIATORS OF RNA INTERFERENCE” filed onMar. 30, 2001 now abandoned, which is herein incorporated by referencein its entirety. Application Ser. No. 09/821,832 claims priority under35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/193,594,entitled “RNA SEQUENCE-SPECIFIC MEDIATORS OF RNA INTERFERENCE” filed onMar. 30, 2000, which is herein incorporated by reference in itsentirety. Application Ser. No. 09/821,832 claims priority under 35U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/265,232,entitled “RNA SEQUENCE-SPECIFIC MEDIATORS OF RNA INTERFERENCE” filed onJan. 31, 2001, which is herein incorporated by reference in itsentirety. Application Ser. No. 09/821,832 claims priority under 35U.S.C. §119 to European Application No. 00 126 325.0 filed on Dec. 1,2000, which is herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. GM034277awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.(1998) Nature 391, 806-811). dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function. RNAi involvesmRNA degradation, but many of the biochemical mechanisms underlying thisinterference are unknown. The recapitulation of the essential featuresof RNAi in vitro is needed for a biochemical analysis of the phenomenon.

SUMMARY OF THE INVENTION

Described herein is gene-specific, dsRNA-mediated interference in acell-free system derived from syncytial blastoderm Drosophila embryos.The in vitro system complements genetic approaches to dissecting themolecular basis of RNAi. As described herein, the molecular mechanismsunderlying RNAi were examined using the Drosophila in vitro system.Results showed that RNAi is ATP-dependent yet uncoupled from mRNAtranslation. That is, protein synthesis is not required for RNAi invitro. In the RNAi reaction, both strands (sense and antisense) of thedsRNA are processed to small RNA fragments or segments of from about 21to about 23 nucleotides (nt) in length (RNAs with mobility in sequencinggels that correspond to markers that are 21-23 nt in length, optionallyreferred to as 21-23 nt RNA). Processing of the dsRNA to the small RNAfragments does not require the targeted mRNA, which demonstrates thatthe small RNA species is generated by processing of the dsRNA and not asa product of dsRNA-targeted mRNA degradation. The mRNA is cleaved onlywithin the region of identity with the dsRNA. Cleavage occurs at sites21-23 nucleotides apart, the same interval observed for the dsRNAitself, suggesting that the 21-23 nucleotide fragments from the dsRNAare guiding mRNA cleavage. That purified 21-23 nt RNAs mediate RNAiconfirms that these fragments are guiding mRNA cleavage.

Accordingly, the present invention relates to isolated RNA molecules(double-stranded; single-stranded) of from about 21 to about 23nucleotides which mediate RNAi. That is, the isolated RNAs of thepresent invention mediate degradation of mRNA of a gene to which themRNA corresponds (mediate degradation of mRNA that is thetranscriptional product of the gene, which is also referred to as atarget gene). For convenience, such mRNA is also referred to herein asmRNA to be degraded. As used herein, the terms RNA, RNA molecule(s), RNAsegment(s) and RNA fragment(s) are used interchangeably to refer to RNAthat mediates RNA interference. These terms include double-stranded RNA,single-stranded RNA, isolated RNA (partially purified RNA, essentiallypure RNA, synthetic RNA, recombinantly produced RNA), as well as alteredRNA that differs from naturally occurring RNA by the addition, deletion,substitution and/or alteration of one or more nucleotides. Suchalterations can include addition of non-nucleotide material, such as tothe end(s) of the 21-23 nt RNA or internally (at one or more nucleotidesof the RNA). Nucleotides in the RNA molecules of the present inventioncan also comprise non-standard nucleotides, including non-naturallyoccurring nucleotides or deoxyribonucleotides. Collectively, all suchaltered RNAs are referred to as analogs or analogs ofnaturally-occurring RNA. RNA of 21-23 nucleotides of the presentinvention need only be sufficiently similar to natural RNA that it hasthe ability to mediate (mediates) RNAi. As used herein the phrase“mediates RNAi” refers to (indicates) the ability to distinguish whichRNAs are to be degraded by the RNAi machinery or process. RNA thatmediates RNAi interacts with the RNAi machinery such that it directs themachinery to degrade particular mRNAs. In one embodiment, the presentinvention relates to RNA molecules of about 21 to about 23 nucleotidesthat direct cleavage of specific mRNA to which their sequencecorresponds. It is not necessary that there be perfect correspondence ofthe sequences, but the correspondence must be sufficient to enable theRNA to direct RNAi cleavage of the target mRNA. In a particularembodiment, the 21-23 nt RNA molecules of the present invention comprisea 3′ hydroxyl group.

The present invention also relates to methods of producing RNA moleculesof about 21 to about 23 nucleotides with the ability to mediate RNAicleavage. In one embodiment, the Drosophila in vitro system is used. Inthis embodiment, dsRNA is combined with a soluble extract derived fromDrosophila embryo, thereby producing a combination. The combination ismaintained under conditions in which the dsRNA is processed to RNAmolecules of about 21 to about 23 nucleotides. In another embodiment,the Drosophila in vitro system is used to obtain RNA sequences of about21 to about 23 nucleotides which mediate RNA interference of the mRNA ofa particular gene (e.g., oncogene, viral gene). In this embodiment,double-stranded RNA that corresponds to a sequence of the gene to betargeted is combined with a soluble extract derived from Drosophilaembryo, thereby producing a combination. The combination is maintainedunder conditions in which the double-stranded RNA is processed to RNA ofabout 21 to about 23 nucleotides in length. As shown herein, 21-23 ntRNA mediates RNAi of the mRNA of the targeted gene (the gene whose mRNAis to be degraded). The method of obtaining 21-23 nt RNAs using theDrosophila in vitro system can further comprise isolating the RNAsequence from the combination.

The present invention also relates to 21-23 nt RNA produced by themethods of the present invention, as well as to 21-23 nt RNAs, producedby other methods, such as chemical synthesis or recombinant DNAtechniques, that have the same or substantially the same sequences asnaturally-occurring RNAs that mediate RNAi, such as those produced bythe methods of the present invention. All of these are referred to as21-23 nt RNAs that mediate RNA interference. As used herein, the termisolated RNA includes RNA obtained by any means, including processing orcleavage of dsRNA as described herein; production by chemical syntheticmethods; and production by recombinant DNA techniques. The inventionfurther relates to uses of the 21-23 nt RNAs, such as for therapeutic orprophylactic treatment and compositions comprising 21-23 nt RNAs thatmediate RNAi, such as pharmaceutical compositions comprising 21-23 ntRNAs and an appropriate carrier (e.g., a buffer or water).

The present invention also relates to a method of mediating RNAinterference of mRNA of a gene in a cell or organism (e.g., mammal suchas a mouse or a human). In one embodiment, RNA of about 21 to about 23nt which targets the mRNA to be degraded is introduced into the cell ororganism. The cell or organism is maintained under conditions underwhich degradation of the mRNA occurs, thereby mediating RNA interferenceof the mRNA of the gene in the cell or organism. The cell or organismcan be one in which RNAi occurs as the cell or organism is obtained or acell or organism can be one that has been modified so that RNAi occurs(e.g., by addition of components obtained from a cell or cell extractthat mediate RNAi or activation of endogenous components). As usedherein, the term “cell or organism in which RNAi occurs” includes both acell or organism in which RNAi occurs as the cell or organism isobtained, or a cell or organism that has been modified so that RNAioccurs. In another embodiment, the method of mediating RNA interferenceof a gene in a cell comprises combining double-stranded RNA thatcorresponds to a sequence of the gene with a soluble extract derivedfrom Drosophila embryo, thereby producing a combination. The combinationis maintained under conditions in which the double-stranded RNA isprocessed to RNAs of about 21 to about 23 nucleotides. 21 to 23 nt RNAis then isolated and introduced into the cell or organism. The cell ororganism is maintained under conditions in which degradation of mRNA ofthe gene occurs, thereby mediating RNA interference of the gene in thecell or organism. As described for the previous embodiment, the cell ororganism is one in which RNAi occurs naturally (in the cell or organismas obtained) or has been modified in such a manner that RNAi occurs. 21to 23 nt RNAs can also be produced by other methods, such as chemicalsynthetic methods or recombinant DNA techniques.

The present invention also relates to biochemical components of a cell,such as a Drosophila cell, that process dsRNA to RNA of about 21 toabout 23 nucleotides. In addition, biochemical components of a cell thatare involved in targeting of mRNA by RNA of about 21 to about 23nucleotides are the subject of the present invention. In bothembodiments, the biochemical components can be obtained from a cell inwhich they occur or can be produced by other methods, such as chemicalsynthesis or recombinant DNA methods. As used herein, the term“isolated” includes materials (e.g., biochemical components, RNA)obtained from a source in which they occur and materials produced bymethods such as chemical synthesis or recombinant nucleic acid (DNA,RNA) methods.

The present invention also relates to a method for knocking down(partially or completely) the targeted gene, thus providing analternative to presently available methods of knocking down (or out) agene or genes. This method of knocking down gene expression can be usedtherapeutically or for research (e.g., to generate models of diseasestates, to examine the function of a gene, to assess whether an agentacts on a gene, to validate targets for drug discovery). In thoseinstances in which gene function is eliminated, the resulting cell ororganism can also be referred to as a knockout. One embodiment of themethod of producing knockdown cells and organisms comprises introducinginto a cell or organism in which a gene (referred to as a targeted gene)is to be knocked down, RNA of about 21 to about 23 nt that targets thegene and maintaining the resulting cell or organism under conditionsunder which RNAi occurs, resulting in degradation of the mRNA of thetargeted gene, thereby producing knockdown cells or organisms. Knockdowncells and organisms produced by the present method are also the subjectof this invention.

The present invention also relates to a method of examining or assessingthe function of a gene in a cell or organism. In one embodiment, RNA ofabout 21 to about 23 nt which targets mRNA of the gene for degradationis introduced into a cell or organism in which RNAi occurs. The cell ororganism is referred to as a test cell or organism. The test cell ororganism is maintained under conditions under which degradation of mRNAof the gene occurs. The phenotype of the test cell or organism is thenobserved and compared to that of an appropriate control cell ororganism, such as a corresponding cell or organism that is treated inthe same manner except that the targeted (specific) gene is nottargeted. A 21 to 23 nt RNA that does not target the mRNA fordegradation can be introduced into the control cell or organism in placeof the RNA introduced into the test cell or organism, although it is notnecessary to do so. A difference between the phenotypes of the test andcontrol cells or organisms provides information about the function ofthe degraded mRNA. In another embodiment, double-stranded RNA thatcorresponds to a sequence of the gene is combined with a soluble extractthat mediates RNAi, such as the soluble extract derived from Drosophilaembryo described herein, under conditions in which the double-strandedRNA is processed to generate RNA of about 21 to about 23 nucleotides.The RNA of about 21 to about 23 nucleotides is isolated and thenintroduced into a cell or organism in which RNAi occurs (test cell ortest organism). The test cell or test organism is maintained underconditions under which degradation of the mRNA occurs. The phenotype ofthe test cell or organism is then observed and compared to that of anappropriate control, such as a corresponding cell or organism that istreated in the same manner as the test cell or organism except that thetargeted gene is not targeted. A difference between the phenotypes ofthe test and control cells or organisms provides information about thefunction of the targeted gene. The information provided may besufficient to identify (define) the function of the gene or may be usedin conjunction with information obtained from other assays or analysesto do so.

Also the subject of the present invention is a method of validatingwhether an agent acts on a gene. In this method, RNA of from about 21 toabout 23 nucleotides that targets the mRNA to be degraded is introducedinto a cell or organism in which RNAi occurs. The cell or organism(which contains the introduced RNA) is maintained under conditions underwhich degradation of mRNA occurs, and the agent is introduced into thecell or organism. Whether the agent has an effect on the cell ororganism is determined; if the agent has no effect on the cell ororganism, then the agent acts on the gene.

The present invention also relates to a method of validating whether agene product is a target for drug discovery or development. RNA of fromabout 21 to about 23 nucleotides that targets the mRNA that correspondsto the gene for degradation is introduced into a cell or organism. Thecell or organism is maintained under conditions in which degradation ofthe mRNA occurs, resulting in decreased expression of the gene. Whetherdecreased expression of the gene has an effect on the cell or organismis determined, wherein if decreased expression of the gene has aneffect, then the gene product is a target for drug discovery ordevelopment.

The present invention also encompasses a method of treating a disease orcondition associated with the presence of a protein in an individualcomprising administering to the individual RNA of from about 21 to about23 nucleotides which targets the mRNA of the protein (the mRNA thatencodes the protein) for degradation. As a result, the protein is notproduced or is not produced to the extent it would be in the absence ofthe treatment.

Also encompassed by the present invention is a gene identified by thesequencing of endogenous 21 to 23 nucleotide RNA molecules that mediateRNA interference.

Also encompassed by the present invention is a method of identifyingtarget sites within an mRNA that are particularly suitable for RNAi aswell as a method of assessing the ability of 21-23 nt RNAs to mediateRNAi.

BRIEF DESCRIPTION OF DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is a schematic representation of reporter mRNAs and dsRNAs Rr-Lucand Pp-Luc. Lengths and positions of the ssRNA, asRNA, and dsRNAs areshown as black bars relative to the Rr-Luc and Pp-Luc reporter mRNAsequences. Black rectangles indicate the two unrelated luciferase codingsequences, lines correspond to the 5′ and 3′ untranslated regions of themRNAs. FIG. 1 discloses “A₂₅” as SEQ ID NO: 20.

FIG. 2A is a graph of the ratio of luciferase activities after targeting50 pM Pp-Luc mRNA with 10 nM ssRNA, asRNA, or dsRNA from the 505 bpsegment of the Pp-Luc gene showing gene-specific interference by dsRNAin vitro. The data are the average values of seven trials±standarddeviation. Four independently prepared lysates were used. Luciferaseactivity was normalized to the buffer control; a ratio equal to oneindicates no gene-specific interference.

FIG. 2B is a graph of the ratio of luciferase activities after targeting50 pM Rr-Luc mRNA with 10 nM ssRNA, asRNA, or dsRNA from the 501 bpsegment of the Rr-Luc gene showing gene-specific interference by dsRNAin vitro. The data are the average values of six trials±standarddeviation. A Rr-Luc/Pp-Luc ratio equal to one indicates no gene-specificinterference.

FIG. 3A is a schematic representation of the experimental strategy usedto show that incubation in the Drosophila embryo lysate potentiatesdsRNA for gene-specific interference. The same dsRNAs used in FIG. 2 (orbuffer) was serially preincubated using two-fold dilutions in sixsuccessive reactions with Drosophila embryo lysate, then tested for itscapacity to block mRNA expression. As a control, the same amount ofdsRNA (10 nM) or buffer was diluted directly in buffer and incubatedwith Pp-Luc and Rr-Luc mRNAs and lysate.

FIG. 3B is a graph of potentiation when targeting Pp-Luc mRNA. Blackcolumns indicate the dsRNA or the buffer was serially preincubated;white columns correspond to a direct 32-fold dilution of the dsRNA.Values were normalized to those of the buffer controls.

FIG. 3C is a graph of potentiation when targeting Rr-Luc mRNA. Thecorresponding buffer control is shown in FIG. 3B.

FIG. 4 is a graph showing effect of competitor dsRNA on gene-specificinterference. Increasing concentrations of nanos dsRNA (508 bp) wereadded to reactions containing 5 nM dsRNA (the same dsRNAs used in FIGS.2A and 2B) targeting Pp-Luc mRNA (black columns, left axis) or Rr-LucmRNA (white columns, right axis). Each reaction contained both a targetmRNA (Pp-Luc for the black columns, Rr-Luc for the white) and anunrelated control mRNA (Rr-Luc for the black columns, Pp-Luc for thewhite). Values were normalized to the buffer control (not shown). Thereactions were incubated under standard conditions (see Methods).

FIG. 5A is a graph showing the effect of dsRNA on mRNA stability.Circles, Pp-Luc mRNA; squares, Rr-Luc mRNA; filled symbols, bufferincubation; open symbols, incubation with Pp-dsRNA.

FIG. 5B is a graph showing the stability of Rr-Luc mRNA incubated withRr-dsRNA or Pp-dsRNA. Filled squares, buffer; open squares, Pp-dsRNA (10nM); open circles, Rr-dsRNA (10 nM).

FIG. 5C is a graph showing the dependence on dsRNA length. The stabilityof the Pp-Luc mRNA was assessed after incubation in lysate in thepresence of buffer or dsRNAs of different lengths. Filled squares,buffer; open circles, 49 bp dsRNA (10 nM); open inverted triangles, 149bp dsRNA (10 nM); open triangles, 505 bp dsRNA (10 nM); open diamonds,997 bp dsRNA (10 nM). Reactions were incubated under standard conditions(see Methods).

FIG. 6 is a graph showing that RNAi Requires ATP. Creatine kinase (CK)uses creatine phosphate (CP) to regenerate ATP. Circles, +ATP, +CP, +CK;squares, −ATP, +CP, +CK; triangles, −ATP, −CP, +CK; inverted triangles,−ATP, +CP, −CK.

FIG. 7A is a graph of protein synthesis, as reflected by luciferaseactivity produced after incubation of Rr-luc mRNA in the in vitro RNAireaction for 1 hour, in the presence of the protein synthesis inhibitorsanisomycin, cycloheximide, or chloramphenicol, relative to a reactionwithout any inhibitor showing that RNAi does not require mRNAtranslation.

FIG. 7B is a graph showing translation of 7-methyl-guanosine- andadenosine-capped Pp-luc mRNAs (circles and squares, respectively) in theRNAi reaction in the absence of dsRNA, as measured by luciferaseactivity produced in a one-hour incubation.

FIG. 7C is a graph showing incubation in an RNAi reaction of uniformly³²P-radiolabeled 7-methyl-guanosine-capped Pp-luc mRNA (circles) andadenosine-capped Pp-luc mRNA (squares), in the presence (open symbols)and absence (filled symbols) of 505 bp Pp-luc dsRNA.

FIG. 8A is a graph of the of the denaturing agarose-gel analysis ofPp-luc mRNA incubated in a standard RNAi reaction with buffer, 505 ntPp-asRNA, or 505 bp Pp-dsRNA for the times indicated showing that asRNAcauses a small amount of RNAi in vitro.

FIG. 8B is a graph of the of the denaturing agarose-gel analysis ofRr-luc mRNA incubated in a standard RNAi reaction with buffer, 505 ntPp-asRNA, or 505 bp Pp-dsRNA for the times indicated showing that asRNAcauses a small amount of RNAi in vitro.

FIG. 9 is a schematic of the positions of the three dsRNAs, ‘A,’‘B,’ and‘C,’ relative to the Rr-luc mRNA. FIG. 9 discloses “A₂₅” as SEQ ID NO:20.

FIG. 10 indicates the cleavage sites mapped onto the first 267 nt of theRr-luc mRNA (SEQ ID NO: 1). The blue bar below the sequence indicatesthe position of dsRNA ‘C,’ and blue circles indicate the position ofcleavage sites caused by this dsRNA. The green bar denotes the positionof dsRNA ‘B,’ and green circles, the cleavage sites. The magenta barindicates the position of dsRNA ‘A,’ and magenta circles, the cleavages.An exceptional cleavage within a run of 7 uracils is marked with a redarrowhead.

FIG. 11 is a proposed model for RNAi. RNAi is envisioned to begin withcleavage of the dsRNA to 21-23 nt products by a dsRNA-specific nuclease,perhaps in a multiprotein complex. These short dsRNAs might then bedissociated by an ATP-dependent helicase, possibly a component of theinitial complex, to 21-23 nt asRNAs that could then target the mRNA forcleavage. The short asRNAs are imagined to remain associated with theRNAi-specific proteins (circles) that were originally bound by thefull-length dsRNA, thus explaining the inefficiency of asRNA to triggerRNAi in vivo and in vitro. Finally, a nuclease (triangles) would cleavethe mRNA.

FIG. 12 is a bar graph showing sequence-specific gene silencing by 21-23nt fragments. Ratio of luciferase activity after targeting of Pp-Luc andRr-Luc mRNA by 5 nM Pp-Luc or Rr-Luc dsRNA (500 bp) or 21-23 ntfragments isolated from a previous incubation of the respective dsRNA inDrosophila lysate. The amount of isolated 21-23 mers present in theincubation reaction correspond to approximately the same amount of 21-23mers generated during an incubation reaction with 5 nM 500 bp dsRNA. Thedata are average values of 3 trials and the standard deviation is givenby error bars. Luciferase activity was normalized to the buffer control.

FIG. 13A illustrates the purification of RNA fragments on a Superdex HR200 10/30 gel filtration column (Pharmacia) using the method describedin Example 4. dsRNA was 32P-labeled, and the radioactivity recovered ineach column fraction is graphed. The fractions were also analyzed bydenaturing gel electrophoresis (inset).

FIG. 13B demonstrates the ability of the Rr-luciferase RNA, afterincubation in the Drosophila lysate and fractionation as in FIG. 13A, tomediate sequence-specific interference with the expression of aRr-luciferase target mRNA. One microliter of each resuspended fractionwas tested in a 10 microliter in vitro RNAi reaction (see Example 1).This procedure yields a concentration of RNA in the standard in vitroRNAi reaction that is approximately equal to the concentration of thatRNA species in the original reaction prior to loading on the column.Relative luminescence per second has been normalized to the averagevalue of the two buffer controls.

FIG. 13C is the specificity control for FIG. 13B. It demonstrates thatthe fractionated RNA of FIG. 13B does not efficiently mediatesequence-specific interference with the expression of a Pp-luciferasemRNA. Assays are as in FIG. 13B.

FIGS. 14A and 14B are schematic representations of reporter constructsand siRNA duplexes. FIG. 14A illustrates the firefly (Pp-luc) and seapansy (Rr-luc) luciferase reporter gene regions from plasmidspGL2-Control, pGL3-Control, and pRL-TK (Promega). SV40 regulatoryelements, the HSV thymidine kinase promoter, and two introns (lines) areindicated. The sequence of GL3 luciferase is 95% identical to GL2, butRL is completely unrelated to both. Luciferase expression from pGL2 isapproximately to 10-fold lower than from pGL3 in transfected mammaliancells. The region targeted by the siRNA duplexes is indicated as blackbar below the coding region of the luciferase genes. FIG. 14B shows thesense (top) and antisense (bottom) sequences of the siRNA duplexestargeting GL2 (SEQ ID Nos: 10 and 11), GL3 (SEQ ID Nos: 12 and 13), andRL (SEQ ID Nos: 14 and 15) luciferase are shown. The GL2 and GL3 siRNAduplexes differ by only 3 single nucleotide substitutions (boxed ingray). As unspecific control, a duplex with the inverted GL2 sequence,invGL2 (SEQ ID Nos: 16 and 17), was synthesized. The 2 nt 3′ overhang of2′-deoxythymidine is indicated as TT; uGL2 (SEQ ID Nos: 18 and 19) issimilar to GL2 siRNA but contains ribo-uridine 3′ overhangs.

FIGS. 15A-15J are graphs showing RNA interference by siRNA duplexes.Ratios of target to control luciferase were normalized to a buffercontrol (bu, black bars); gray bars indicate ratios of Photinus pyralis(Pp-luc) GL2 or GL3 luciferase to Renilla reniformis (Rr-luc) RLluciferase (left axis), white bars indicate RL to GL2 or GL3 ratios(right axis). FIGS. 15A, 15C, 15E, 15G, and 15I show results ofexperiments performed with the combination of pGL2-Control and pRL-TKreporter plasmids, FIGS. 15B, 15D, 15F, 15H, and 15J with pGL3-Controland pRL-TK reporter plasmids. The cell line used for the interferenceexperiment is indicated at the top of each plot. The ratios ofPp-luc/Rr-luc for the buffer control (bu) varied between 0.5 and 10 forpGL2/pRL, and between 0.03 and 1 for pGL3/pRL, respectively, beforenormalization and between the various cell lines tested. The plotteddata were averaged from three independent experiments±S.D. FIGS. 16A-16Fare graphs showing the effects of 21 nt siRNAs, 50 bp, and 500 bp dsRNAson luciferase expression in HeLa cells. The exact length of the longdsRNAs is indicated below the bars. FIGS. 16A, 16C, and 16E describeexperiments performed with pGL2-Control and pRL-TK reporter plasmids,FIGS. 16B, 16D, and 16F with pGL3-Control and pRL-TK reporter plasmids.The data were averaged from two independent experiments±S.D. FIGS. 16A,16B, Absolute Pp-luc expression, plotted in arbitrary luminescenceunits. FIG. 16C, 16D, Rr-luc expression, plotted in arbitraryluminescence units. FIGS. 16E, 16F, Ratios of normalized target tocontrol luciferase. The ratios of luciferase activity for siRNA duplexeswere normalized to a buffer control (bu, black bars); the luminescenceratios for 50 or 500 bp dsRNAs were normalized to the respective ratiosobserved for 50 and 500 bp dsRNA from humanized GFP (hG, black bars). Itshould be noted, that the overall differences in sequence between the 49and 484 bp dsRNAs targeting GL2 and GL3 are not sufficient to conferspecificity between GL2 and GL3 targets (43 nt uninterrupted identity in49 bp segment, 239 nt longest uninterrupted identity in 484 bp segment)(Parrish, S., et al., Mol. Cell, 6:1077-1087 (2000)).

DETAILED DESCRIPTION OF THE INVENTION

Double-stranded (dsRNA) directs the sequence-specific degradation ofmRNA through a process known as RNA interference (RNAi). The process isknown to occur in a wide variety of organisms, including embryos ofmammals and other vertebrates. Using the Drosophila in vitro systemdescribed herein, it has been demonstrated that dsRNA is processed toRNA segments 21-23 nucleotides (nt) in length, and furthermore, thatwhen these 21-23 nt fragments are purified and added back to Drosophilaextracts, they mediate RNA interference in the absence of longer dsRNA.Thus, these 21-23 nt fragments are sequence-specific mediators of RNAdegradation. A molecular signal, which may be the specific length of thefragments, must be present in these 21-23 nt fragments to recruitcellular factors involved in RNAi. This present invention encompassesthese 21-23 nt fragments and their use for specifically inactivatinggene function. The use of these fragments (or recombinantly produced orchemically synthesized oligonucleotides of the same or similar nature)enables the targeting of specific mRNAs for degradation in mammaliancells. Use of long dsRNAs in mammalian cells to elicit RNAi is usuallynot practical, presumably because of the deleterious effects of theinterferon response. Specific targeting of a particular gene function,which is possible with 21-23 nt fragments of the present invention, isuseful in functional genomic and therapeutic applications.

In particular, the present invention relates to RNA molecules of about21 to about 23 nucleotides that mediate RNAi. In one embodiment, thepresent invention relates to RNA molecules of about 21 to about 23nucleotides that direct cleavage of specific mRNA to which theycorrespond. The 21-23 nt RNA molecules of the present invention can alsocomprise a 3′ hydroxyl group. The 21-23 nt RNA molecules can besingle-stranded or double stranded (as two 21-23 nt RNAs); suchmolecules can be blunt ended or comprise overhanging ends (e.g., 5′,3′). In specific embodiments, the RNA molecule is double stranded andeither blunt ended or comprises overhanging ends (as two 21-23 nt RNAs).

In one embodiment, at least one strand of the RNA molecule has a 3′overhang from about 1 to about 6 nucleotides (e.g., pyrimidinenucleotides, purine nucleotides) in length. In other embodiments, the 3′overhang is from about 1 to about 5 nucleotides, from about 1 to about 3nucleotides and from about 2 to about 4 nucleotides in length. In oneembodiment the RNA molecule is double stranded, one strand has a 3′overhang and the other strand can be blunt-ended or have an overhang. Inthe embodiment in which the RNA molecule is double stranded and bothstrands comprise an overhang, the length of the overhangs may be thesame or different for each strand. In a particular embodiment, the RNAof the present invention comprises 21 nucleotide strands which arepaired and which have overhangs of from about 1 to about 3, particularlyabout 2, nucleotides on both 3′ ends of the RNA. In order to furtherenhance the stability of the RNA of the present invention, the 3′overhangs can be stabilized against degradation. In one embodiment, theRNA is stabilized by including purine nucleotides, such as adenosine orguanosine nucleotides. Alternatively, substitution of pyrimidinenucleotides by modified analogues, e.g., substitution of uridine 2nucleotide 3′ overhangs by 2′-deoxythymidine is tolerated and does notaffect the efficiency of RNAi. The absence of a 2′ hydroxylsignificantly enhances the nuclease resistance of the overhang in tissueculture medium.

The 21-23 nt RNA molecules of the present invention can be obtainedusing a number of techniques known to those of skill in the art. Forexample, the RNA can be chemically synthesized or recombinantly producedusing methods known in the art. The 21-23 nt RNAs can also be obtainedusing the Drosophila in vitro system described herein. Use of theDrosophila in vitro system entails combining dsRNA with a solubleextract derived from Drosophila embryo, thereby producing a combination.The combination is maintained under conditions in which the dsRNA isprocessed to RNA of about 21 to about 23 nucleotides. The Drosophila invitro system can also be used to obtain RNA of about 21 to about 23nucleotides in length which mediates RNA interference of the mRNA of aparticular gene (e.g., oncogene, viral gene). In this embodiment,double-stranded RNA that corresponds to a sequence of the gene iscombined with a soluble extract derived from Drosophila embryo, therebyproducing a combination. The combination is maintained under conditionsin which the double-stranded RNA is processed to the RNA of about 21 toabout 23 nucleotides. As shown herein, 21-23 nt RNA mediates RNAi of themRNA to be degraded. The present invention also relates to the 21-23 ntRNA molecules produced by the methods described herein.

In one embodiment, the methods described herein are used to identify orobtain 21-23 nt RNA molecules that are useful as sequence-specificmediators of RNA degradation and, thus, for inhibiting mRNAs, such ashuman mRNAs, that encode products associated with or causative of adisease or an undesirable condition. For example, production of anoncoprotein or viral protein can be inhibited in humans in order toprevent the disease or condition from occurring, limit the extent towhich it occurs or reverse it. If the sequence of the gene to betargeted in humans is known, 21-23 nt RNAs can be produced and testedfor their ability to mediate RNAi in a cell, such as a human or otherprimate cell. Those 21-23 nt human RNA molecules shown to mediate RNAican be tested, if desired, in an appropriate animal model to furtherassess their in vivo effectiveness. Additional copies of 21-23 nt RNAsshown to mediate RNAi can be produced by the methods described herein.

The method of obtaining the 21-23 nt RNA sequence using the Drosophilain vitro system can further comprise isolating the RNA sequence from thecombination. The 21-23 nt RNA molecules can be isolated using a numberof techniques known to those of skill in the art. For example, gelelectrophoresis can be used to separate 21-23 nt RNAs from thecombination, gel slices comprising the RNA sequences removed and RNAseluted from the gel slices. Alternatively, non-denaturing methods, suchas non-denaturing column chromatography, can be used to isolate the RNAproduced. In addition, chromatography (e.g., size exclusionchromatography), glycerol gradient centrifugation, affinity purificationwith antibody can be used to isolate 21-23 nt RNAs. The RNA-proteincomplex isolated from the Drosophila in vitro system can also be useddirectly in the methods described herein (e.g., method of mediating RNAiof mRNA of a gene). Soluble extracts derived from Drosophila embryo thatmediate or RNAi are encompassed by the invention. The soluble Drosophilaextract can be obtained in a variety of ways. For example, the solubleextract can be obtained from syncytial blastoderm Drosophila embryos asdescribed in Examples 1, 2, and 3. Soluble extracts can be derived fromother cells in which RNAi occurs. Alternatively, soluble extracts can beobtained from a cell that does not carry out RNAi. In this instance, thefactors needed to mediate RNAi can be introduced into such a cell andthe soluble extract is then obtained. The components of the extract canalso be chemically synthesized and/or combined using methods known inthe art.

Any dsRNA can be used in the methods of the present invention, providedthat it has sufficient homology to the targeted gene to mediate RNAi.The sequence of the dsRNA for use in the methods of the presentinvention need not be known. Alternatively, the dsRNA for use in thepresent invention can correspond to a known sequence, such as that of anentire gene (one or more) or portion thereof. There is no upper limit onthe length of the dsRNA that can be used. For example, the dsRNA canrange from about 21 base pairs (bp) of the gene to the full length ofthe gene or more. In one embodiment, the dsRNA used in the methods ofthe present invention is about 1000 bp in length. In another embodiment,the dsRNA is about 500 bp in length. In yet another embodiment, thedsRNA is about 22 bp in length.

The 21 to 23 nt RNAs described herein can be used in a variety of ways.For example, the 21 to 23 nt RNA molecules can be used to mediate RNAinterference of mRNA of a gene in a cell or organism. In a specificembodiment, the 21 to 23 nt RNA is introduced into human cells or ahuman in order to mediate RNA interference in the cells or in cells inthe individual, such as to prevent or treat a disease or undesirablecondition. In this method, a gene (or genes) that cause or contribute tothe disease or undesirable condition is targeted and the correspondingmRNA (the transcriptional product of the targeted gene) is degraded byRNAi. In this embodiment, an RNA of about 21 to about 23 nucleotidesthat targets the corresponding mRNA (the mRNA of the targeted gene) fordegradation is introduced into the cell or organism. The cell ororganism is maintained under conditions under which degradation of thecorresponding mRNA occurs, thereby mediating RNA interference of themRNA of the gene in the cell or organism. In a particular embodiment,the method of mediating RNA interference of a gene in a cell comprisescombining double-stranded RNA that corresponds to a sequence of the genewith a soluble extract derived from Drosophila embryo, thereby producinga combination. The combination is maintained under conditions in whichthe double-stranded RNA is processed to RNA of about 21 to about 23nucleotides. The 21 to 23 nt RNA is then isolated and introduced intothe cell or organism. The cell or organism is maintained underconditions in which degradation of mRNA of the gene occurs, therebymediating RNA interference of the gene in the cell or organism. In theevent that the 21-23 nt RNA is introduced into a cell in which RNAi,does not normally occur, the factors needed to mediate RNAi areintroduced into such a cell or the expression of the needed factors isinduced in such a cell. Alternatively, 21 to 23 nt RNA produced by othermethods (e.g., chemical synthesis, recombinant DNA production) to have acomposition the same as or sufficiently similar to a 21 to 23 nt RNAknown to mediate RNAi can be similarly used to mediate RNAi. Such 21 to23 nt RNAs can be altered by addition, deletion, substitution ormodification of one or more nucleotides and/or can comprisenon-nucleotide materials. A further embodiment of this invention is anex vivo method of treating cells from an individual to degrade a gene(s)that causes or is associated with a disease or undesirable condition,such as leukemia or AIDS. In this embodiment, cells to be treated areobtained from the individual using known methods (e.g., phlebotomy orcollection of bone marrow) and 21-23 nt RNAs that mediate degradation ofthe corresponding mRNA(s) are introduced into the cells, which are thenre-introduced into the individual. If necessary, biochemical componentsneeded for RNAi to occur can also be introduced into the cells.

The mRNA of any gene can be targeted for degradation using the methodsof mediating interference of mRNA described herein. For example, anycellular or viral mRNA, can be targeted, and, as a result, the encodedprotein (e.g., an oncoprotein, a viral protein), expression will bediminished. In addition, the mRNA of any protein associatedwith/causative of a disease or undesirable condition can be targeted fordegradation using the methods described herein.

The present invention also relates to a method of examining the functionof a gene in a cell or organism. In one embodiment, an RNA sequence ofabout 21 to about 23 nucleotides that targets mRNA of the gene fordegradation is introduced into the cell or organism. The cell ororganism is maintained under conditions under which degradation of mRNAof the gene occurs. The phenotype of the cell or organism is thenobserved and compared to an appropriate control, thereby providinginformation about the function of the gene. In another embodiment,double-stranded RNA that corresponds to a sequence of the gene iscombined with a soluble extract derived from Drosophila embryo underconditions in which the double-stranded RNA is processed to generate RNAof about 21 to about 23 nucleotides. The RNA of about 21 to about 23nucleotides is isolated and then introduced into the cell or organism.The cell or organism is maintained under conditions in which degradationof the mRNA of the gene occurs. The phenotype of the cell or organism isthen observed and compared to an appropriate control, therebyidentifying the function of the gene.

A further aspect of this invention is a method of assessing the abilityof 21-23 nt RNAs to mediate RNAi and, particularly, determining which21-23 nt RNA(s) most efficiently mediate RNAi. In one embodiment of themethod, dsRNA corresponding to a sequence of an mRNA to be degraded iscombined with detectably labeled (e.g., end-labeled, such asradiolabeled) mRNA and the soluble extract of this invention, therebyproducing a combination. The combination is maintained under conditionsunder which the double-stranded RNA is processed and the mRNA isdegraded. The sites of the most effective cleavage are mapped bycomparing the migration of the labeled mRNA cleavage products to markersof known length. 21 mers spanning these sites are then designed andtested for their efficiency in mediating RNAi.

Alternatively, the extract of the present invention can be used todetermine whether there is a particular segment or particular segmentsof the mRNA corresponding to a gene which are more efficiently targetedby RNAi than other regions and, thus, can be especially useful targetsites. In one embodiment, dsRNA corresponding to a sequence of a gene tobe degraded, labeled mRNA of the gene is combined with a soluble extractthat mediates RNAi, thereby producing a combination. The resultingcombination is maintained under conditions under which the dsRNA isdegraded and the sites on the mRNA that are most efficiently cleaved areidentified, using known methods, such as comparison to known sizestandards on a sequencing gel.

OVERVIEW OF EXAMPLES

Biochemical analysis of RNAi has become possible with the development ofthe in vitro Drosophila embryo lysate that recapitulates dsRNA-dependentsilencing of gene expression described in Example 1 (Tuschl et al.,Genes Dev., 13:3191-7 (1999)). In the in vitro system, dsRNA, but notsense or asRNA, targets a corresponding mRNA for degradation, yet doesnot affect the stability of an unrelated control mRNA. Furthermore,pre-incubation of the dsRNA in the lysate potentiates its activity fortarget mRNA degradation, suggesting that the dsRNA must be converted toan active form by binding proteins in the extract or by covalentmodification (Tuschl et al., Genes Dev., 13:3191-7 (1999)).

The development of a cell-free system from syncytial blastodermDrosophila embryos that recapitulates many of the features of RNAi isdescribed herein. The interference observed in this reaction issequence-specific, is promoted by dsRNA, but not by single-stranded RNA,functions by specific mRNA degradation, requires a minimum length ofdsRNA and is most efficient with long dsRNA. Furthermore, preincubationof dsRNA potentiates its activity. These results demonstrate that RNAiis mediated by sequence specific processes in soluble reactions.

As described in Example 2, the in vitro system was used to analyze therequirements of RNAi and to determine the fate of the dsRNA and themRNA. RNAi in vitro requires ATP, but does not require either mRNAtranslation or recognition of the 7-methyl-guanosine cap of the targetedmRNA. The dsRNA, but not single-stranded RNA, is processed in vitro to apopulation of 21-23 nt species. Deamination of adenosines within thedsRNA does not appear to be required for formation of the 21-23 nt RNAs.As described herein, the mRNA is cleaved only in the regioncorresponding to the sequence of the dsRNA and that the mRNA is cleavedat 21-23 nt intervals, strongly indicating that the 21-23 nt fragmentsfrom the dsRNA are targeting the cleavage of the mRNA. Furthermore, asdescribed in Examples 3 and 4, when the 21-23 nt fragments are purifiedand added back to the soluble extract, they mediate RNA.

The present invention is illustrated by the following examples, whichare not intended to be limiting in any way.

Example 1 Targeted mRNA Degradation by Double-Stranded RNA In VitroMaterials and Methods

RNAs

Rr-Luc mRNA consisted of the 926 nt Rr luciferase coding sequenceflanked by 25 nt of 5′ untranslated sequence from the pSP64 plasmidpolylinker and 25 nt of 3′ untranslated sequence consisting of 19 nt ofpSP64 plasmid polylinker sequence followed by a 6 nt Sac I site. Pp-LucmRNA contained the 1653 nt Pp luciferase coding sequence with a Kpn Isite introduced immediately before the Pp luciferase stop codon. The Ppcoding sequence was flanked by 5′ untranslated sequences consisting of21 nt of pSP64 plasmid polylinker followed by the 512 nt of the 5′untranslated region (UTR) from the Drosophila hunchback mRNA and 3′untranslated sequences consisting of the 562 nt hunchback 3′ UTRfollowed by a 6 nt Sac I site. The hunchback 3′ UTR sequences usedcontained six G-to-U mutations that disrupt function of the NanosResponse Elements in vivo and in vitro. Both reporter mRNAs terminatedin a 25 nt poly(A) tail (SEQ ID NO: 20) encoded in the transcribedplasmid. For both Rr-Luc and Pp-Luc mRNAs, the transcripts weregenerated by run-off transcription from plasmid templates cleaved at anNsi I site that immediately followed the 25 nt encoded poly(A) tail (SEQID NO: 20). To ensure that the transcripts ended with a poly(A) tail,the Nsi I-cleaved transcription templates were resected with T4 DNAPolymerase in the presence of dNTPs. The SP6 mMessage mMachine kit(Ambion) was used for in vitro transcription. Using this kit, about 80%of the resulting transcripts are 7-methyl guanosine capped.³²P-radiolabeling was accomplished by including α-³²P-UTP in thetranscription reaction.

For Pp-Luc, ss, as, and dsRNA corresponded to positions 93 to 597relative to the start of translation, yielding a 505 bp dsRNA. ForRr-Luc, ss, as, and dsRNA corresponded to positions 118 to 618 relativeto the start of translation, yielding a 501 bp dsRNA. The Drosophilananos competitor dsRNA corresponded to positions 122 to 629 relative tothe start of translation, yielding a 508 bp dsRNA. ssRNA, asRNA, anddsRNA (diagrammed in FIG. 1) were transcribed in vitro with T7 RNApolymerase from templates generated by the polymerase chain reaction.After gel purification of the T7 RNA transcripts, residual DNA templatewas removed by treatment with RQ1 DNase (Promega). The RNA was thenextracted with phenol and chloroform, and then precipitated anddissolved in water.

RNA Annealing and Native Gel Electrophoresis.

ssRNA and asRNA (0.5 μM) in 10 mM Tris-HCl (pH 7.5) with 20 mM NaCl wereheated to 95° C. for 1 min then cooled and annealed at room temperaturefor 12 to 16 h. The RNAs were precipitated and resuspended in lysisbuffer (below). To monitor annealing, RNAs were electrophoresed in a 2%agarose gel in TBE buffer and stained with ethidium bromide (Sambrook etal., Molecular Cloning. Cold Spring Harbor Laboratory Press, Plainview,N.Y. (1989)).

Lysate Preparation

Zero- to two-hour old embryos from Oregon R flies were collected onyeasted molasses agar at 25° C. Embryos were dechorionated for 4 to 5min in 50% (v/v) bleach, washed with water, blotted dry, and transferredto a chilled Potter-Elvehjem tissue grinder (Kontes). Embryos were lysedat 4° C. in one ml of lysis buffer (100 mM potassium acetate, 30 mMHEPES-KOH, pH 7.4, 2 mM magnesium acetate) containing 5 mMdithiothreitol (DTT) and 1 mg/ml Pefabloc SC (Boehringer-Mannheim) pergram of damp embryos. The lysate was centrifuged for 25 min at 14,500×gat 4° C., and the supernatant flash frozen in aliquots in liquidnitrogen and stored at −80° C.

Reaction Conditions

Lysate preparation and reaction conditions were derived from thosedescribed by Hussain and Leibowitz (Hussain and Leibowitz, Gene 46:13-23(1986)). Reactions contained 50% (v/v) lysate, mRNAs (10 to 50 pM finalconcentration), and 10% (v/v) lysis buffer containing the ssRNA, asRNA,or dsRNA (10 nM final concentration). Each reaction also contained 10 mMcreatine phosphate, 10 μg/ml creatine phosphokinase, 100 μM GTP, 100 μMUTP, 100 μM CTP, 500 μM ATP, 5 μM DTT, 0.1 U/mL RNasin (Promega), and100 μM of each amino acid. The final concentration of potassium acetatewas adjusted to 100 mM. For standard conditions, the reactions wereassembled on ice and then pre-incubated at 25° C. for 10 min beforeadding mRNA. After adding mRNAs, the incubation was continued for anadditional 60 min. The 10 min preincubation step was omitted for theexperiments in FIGS. 3A-3C and 5A-5C. Reactions were quenched with fourvolumes of 1.25× Passive Lysis Buffer (Promega). Pp and Rr luciferaseactivity was detected in a Monolight 2010 Luminometer (AnalyticalLuminescence Laboratory) using the Dual-Luciferase Reporter Assay System(Promega).

RNA Stability

Reactions with ³²P-radiolabeled mRNA were quenched by the addition of 40volumes of 2×PK buffer (200 mM Tris-HCl, pH 7.5, 25 mM EDTA, 300 mMNaCl, 2% w/v sodium dodecyl sulfate). Proteinase K (E.M. Merck;dissolved in water) was added to a final concentration of 465 μg/ml. Thereactions were then incubated for 15 min at 65° C., extracted withphenol/chloroform/isoamyl alcohol (25:24:1), and precipitated with anequal volume of isopropanol. Reactions were analyzed by electrophoresisin a formaldehyde/agarose (0.8% w/v) gel (Sambrook et al., MolecularCloning. Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989)).Radioactivity was detected by exposing the agarose gel [dried undervacuum onto Nytran Plus membrane (Amersham)] to an image plate (Fujix)and quantified using a Fujix Bas 2000 and Image Gauge 3.0 (Fujix)software.

Commercial Lysates

Untreated rabbit reticulocyte lysate (Ambion) and wheat germ extract(Ambion) reactions were assembled according to the manufacturer'sdirections. dsRNA was incubated in the lysate at 27° C. (wheat germ) or30° C. (reticulocyte lysate) for 10 min prior to the addition of mRNAs.

Results and Discussion

To evaluate if dsRNA could specifically block gene expression in vitro,reporter mRNAs derived from two different luciferase genes that areunrelated both in sequence and in luciferin substrate specificity wereused: Renilla reniformis (sea pansy) luciferase (Rr-Luc) and Photurispennsylvanica (firefly) luciferase (Pp-Luc). dsRNA generated from onegene was used to target that luciferase mRNA whereas the otherluciferase mRNA was an internal control co-translated in the samereaction. dsRNAs of approximately 500 bp were prepared by transcriptionof polymerase-chain reaction products from the Rr-Luc and Pp-Luc genes.Each dsRNA began ˜100 bp downstream of the start of translation (FIG.1). Sense (ss) and anti-sense (as) RNA were transcribed in vitro andannealed to each other to produce the dsRNA. Native gel electrophoresisof the individual Rr 501 and Pp 505 nt as RNA and ssRNA used to form theRr and Pp dsRNAs was preformed. The ssRNA, asRNA, and dsRNAs were eachtested for their ability to block specifically expression of theircognate mRNA but not the expression of the unrelated internal controlmRNA.

The ssRNA, asRNA, or dsRNA was incubated for 10 min in a reactioncontaining Drosophila embryo lysate, then both Pp-Luc and Rr-Luc mRNAswere added and the incubation continued for an additional 60 min. TheDrosophila embryo lysate efficiently translates exogenously transcribedmRNA under the conditions used. The amounts of Pp-Luc and Rr-Luc enzymeactivities were measured and were used to calculate ratios of eitherPp-Luc/Rr-Luc (FIG. 2A) or Rr-Luc/Pp-Luc (FIG. 2B). To facilitatecomparison of different experiments, the ratios from each experimentwere normalized to the ratio observed for a control in which buffer wasadded to the reaction in place of ssRNA, asRNA, or dsRNA.

FIG. 2A shows that a 10 nM concentration of the 505 bp dsRNA identicalto a portion of the sequence of the Pp-Luc gene specifically inhibitedexpression of the Pp-Luc mRNA but did not affect expression of theRr-Luc internal control. Neither ssRNA nor asRNA affected expression ofPp-Luc or the Rr-Luc internal control. Thus, Pp-Luc expression wasspecifically inhibited by its cognate dsRNA. Conversely, a 10 nMconcentration of the 501 bp dsRNA directed against the Rr-Luc mRNAspecifically inhibited Rr-Luc expression but not that of the Pp-Lucinternal control (FIG. 2B). Again, comparable levels of ssRNA or asRNAhad little or no effect on expression of either reporter mRNA. Onaverage, dsRNA reduced specific luciferase expression by 70% in theseexperiments, in which luciferase activity was measured after 1 hincubation. In other experiments in which the translational capacity ofthe reaction was replenished by the addition of fresh lysate andreaction components, a further reduction in targeted luciferase activityrelative to the internal control was observed.

The ability of dsRNA but not asRNA to inhibit gene expression in theselysates is not merely a consequence of the greater stability of thedsRNA (half-life about 2 h) relative to the single-stranded RNAs(half-life ˜10 min). ssRNA and asRNA transcribed with a 7-methylguanosine cap were as stable in the lysate as uncapped dsRNA, but do notinhibit gene expression. In contrast, dsRNA formed from the capped ssRNAand asRNA specifically blocks expression of the targeted mRNA.

Effective RNAi in Drosophila requires the injection of about 0.2 fmol ofdsRNA into a syncytial blastoderm embryo (Kennerdell and Carthew, Cell95:1017-1026 (1998); Carthew,www1.pitted.edu/˜carthew/manual/RN-Ai_Protocol.html (1999)). Since theaverage volume of a Drosophila embryo is approximately 7.3 nl, thiscorresponds to an intracellular concentration of about 25 nM (Mazur etal., Cryobiology 25:543-544 (1988)). Gene expression in the Drosophilalysate was inhibited by a comparable concentration of dsRNA (10 nM), butlowering the dsRNA concentration ten-fold decreased the amount ofspecific interference. Ten nanomolar dsRNA corresponds to a 200-foldexcess of dsRNA over target mRNA added to the lysate. To test if thisexcess of dsRNA might reflect a time- and/or concentration-dependentstep in which the input dsRNA was converted to a form active forgene-specific interference, the effect of preincubation of the dsRNA onits ability to inhibit expression of its cognate mRNA was examined.Because the translational capacity of the lysates is significantlyreduced after 30 min of incubation at 25° C. (unpublished observations),it was desired to ensure that all factors necessary for RNAi remainedactive throughout the pre-incubation period. Therefore, every 30 min, areaction containing dsRNA and lysate was mixed with a fresh reactioncontaining unincubated lysate (FIG. 3A). After six successive serialtransfers spanning 3 hours of preincubation, the dsRNA, now diluted64-fold relative to its original concentration, was incubated withlysate and 50 pM of target mRNA for 60 min. Finally, the Pp-Luc andRr-Luc enzyme levels were measured. For comparison, the input amount ofdsRNA (10 nM) was diluted 32-fold in buffer, and its capacity togenerate gene-specific dsRNA interference in the absence of anypreincubation step was assessed.

The preincubation of the dsRNA in lysate significantly potentiated itscapacity to inhibit specific gene expression. Whereas the dsRNA diluted32-fold showed no effect, the preincubated dsRNA was, withinexperimental error, as potent as undiluted dsRNA, despite havingundergone a 64-fold dilution. Potentiation of the dsRNA by preincubationwas observed for dsRNAs targeting both the Pp-Luc mRNA (FIG. 3B) and theRr-Luc mRNA (FIG. 3C). Taking into account the 64-fold dilution, theactivation conferred by preincubation allowed a 156 pM concentration ofdsRNA to inhibit 50 pM target mRNA. Further, dilution of the “activated”dsRNA may be effective but has not been tested. We note that althoughboth dsRNAs tested were activated by the preincubation procedure, eachfully retained its specificity to interfere with expression only of themRNA to which it is homologous. Further study of the reactions mayprovide a route to identifying the mechanism of dsRNA potentiation.

One possible explanation for the observation that preincubation of thedsRNA enhances its capacity to inhibit gene expression in these lysatesis that specific factors either modify and/or associate with the dsRNA.Accordingly, the addition of increasing amounts of dsRNA to the reactionmight titrate such factors and decrease the amount of gene-specificinterference caused by a second dsRNA of unrelated sequence. For bothPp-Luc mRNA and Rr-Luc mRNA, addition of increasing concentrations ofthe unrelated Drosophila nanos dsRNA to the reaction decreased theamount of gene-specific interference caused by dsRNA targeting thereporter mRNA (FIG. 4). None of the tested concentrations of nanos dsRNAaffected the levels of translation of the untargeted mRNA, demonstratingthat the nanos dsRNA specifically titrated factors involved ingene-specific interference and not components of the translationalmachinery. The limiting factor(s) was titrated by addition ofapproximately 1000 nM dsRNA, a 200-fold excess over the 5 nM of dsRNAused to produce specific interference.

Interference in vitro might reflect either a specific inhibition of mRNAtranslation or the targeted destruction of the specific mRNA. Todistinguish these two possibilities, the fates of the Pp-Luc and Rr-LucmRNAs were examined directly using ³²P-radiolabeled substrates.Stability of 10 nM Pp-Luc mRNA or Rr-Luc mRNA incubated in lysate witheither buffer or 505 bp Pp-dsRNA (10 nM). Samples were deproteinizedafter the indicated times and the ³²P-radiolabeled mRNAs were thenresolved by denaturing gel electrophoresis. In the absence of dsRNA,both the Pp-Luc and Rr-Luc mRNAs were stable in the lysates, with ˜75%of the input mRNA remaining after 3 h of incubation. (About 25% of theinput mRNA is rapidly degraded in the reaction and likely representsuncapped mRNA generated by the in vitro transcription process.) In thepresence of dsRNA (10 nM, 505 bp) targeting the Pp-Luc mRNA, less than15% of the Pp-Luc mRNA remained after 3 h (FIG. 5A). As expected, theRr-Luc mRNA remained stable in the presence of the dsRNA targetingPp-Luc mRNA. Conversely, dsRNA (10 nM, 501 bp) targeting the Rr-Luc mRNAcaused the destruction of the Rr-Luc mRNA but had no effect on thestability of Pp-Luc mRNA (FIG. 5B). Thus, the dsRNA specifically causedaccelerated decay of the mRNA to which it is homologous with no effecton the stability of the unrelated control mRNA. This finding indicatesthat in vivo, at least in Drosophila, the effect of dsRNA is to directlydestabilize the target mRNA, not to change the subcellular localizationof the mRNA, for example, by causing it to be specifically retained inthe nucleus, resulting in non-specific degradation.

These results are consistent with the observation that RNAi leads toreduced cytoplasmic mRNA levels in vivo, as measured by in situhybridization (Montgomery et al., Proc. Natl. Acad. Sci. USA95:15502-15507 (1998)) and Northern blotting (Ngo et al., Proc. Natl.Acad. Sci. USA 95:14687-14692 (1998)). Northern blot analyses intrypanosomes and hydra suggest that dsRNA typically decreases mRNAlevels by less than 90% (Ngo et al., Proc. Natl. Acad. Sci. USA95:14687-14692 (1998); Lohmann et al., Dev. Biol. 214:211-214 (1999)).The data presented here show that in vitro mRNA levels are reduced 65 to85% after three hours incubation, an effect comparable with observationsin vivo. They also agree with the finding that RNAi in C. elegans ispost-transcriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA95:15502-15507 (1998)). The simplest explanation for the specificeffects on protein synthesis is that it reflects the accelerated rate ofRNA decay. However, the results do not exclude independent but specificeffects on translation as well as stability.

In vivo, RNAi appears to require a minimum length of dsRNA (Ngo et al.,Proc. Natl. Acad. Sci., USA, 95:14687-14692 (1998)). The ability of RNAduplexes of lengths 49 bp, 149 bp, 505 bp, and 997 bp (diagrammed inFIG. 1) to target the degradation of the Pp-Luc mRNA in vitro wasassessed. In good agreement with in vivo observations, the 49 bp dsRNAwas ineffective in vitro, while the 149 bp dsRNA enhanced mRNA decayonly slightly, and both the 505 and 997 bp dsRNAs caused robust mRNAdegradation (FIG. 5C). 50 bp dsRNA targeting other portions of the mRNAcause detectable mRNA degradation, though not as robust as that seen for500 bp dsRNA. Thus, although some short dsRNA do not mediate RNAi,others of approximately the same length, but different composition, willbe able to do so.

Whether the gene-specific interference observed in Drosophila lysateswas a general property of cell-free translation systems was examined.The effects of dsRNAs on expression of Pp-Luc and Rr-Luc mRNA wereexamined in commercially available wheat germ extracts and rabbitreticulocyte lysates. There was no effect of addition of 10 nM of eitherssRNA, asRNA, or dsRNA on the expression of either mRNA reporter inwheat germ extracts. In contrast, the addition of 10 nM of dsRNA to therabbit reticulocyte lysate caused a profound and rapid, non-specificdecrease in mRNA stability. For example, addition of Rr-Luc dsRNA causeddegradation of both Rr-Luc and Pp-Luc mRNAs within 15 min. The samenon-specific effect was observed upon addition of Pp-Luc dsRNA. Thenon-specific destruction of mRNA induced by the addition of dsRNA to therabbit reticulocyte lysate presumably reflects the previously observedactivation of RNase L by dsRNA (Clemens and Williams, Cell 13:565-572(1978); Williams et al., Nucleic Acids Res. 6:1335-1350 (1979); Zhou etal., Cell 72:753-765 (1993); Matthews, Interactions between Viruses andthe Cellular Machinery for Protein Synthesis. In Translational Control(eds. J. Hershey, M. Mathews and N. Sonenberg), pp. 505-548. Cold SpringHarbor Laboratory Press, Plainview, N.Y. (1996)). Mouse cell lineslacking dsRNA-induced anti-viral pathways have recently been described(Zhou et al., Virology 258:435-440 (1999)) and may be useful in thesearch for mammalian RNAi. Although RNAi is known to exist in somemammalian cells (Wianny and Zernicka-Goetz Nat. Cell Biol. 2: 70-75(2000)), in many mammalian cell types its presence is likely obscured bythe rapid induction by dsRNA of non-specific anti-viral responses.

dsRNA-targeted destruction of specific mRNA is characteristic of RNAi,which has been observed in vivo in many organisms, including Drosophila.The system described above recapitulates in a reaction in vitro manyaspects of RNAi. The targeted mRNA is specifically degraded whereasunrelated control mRNAs present in the same solution are not affected.The process is most efficient with dsRNAs greater than 150 bp in length.The dsRNA-specific degradation reaction in vitro is probably general tomany, if not all, mRNAs since it was observed using two unrelated genes.

The magnitude of the effects on mRNA stability in vitro described hereinare comparable with those reported in vivo (Ngo et al., Proc. Natl.Acad. Sci., USA, 95:14687-14692 (1998); Lohmann et al., Dev. Biol.,214:211-214 (1999). However, the reaction in vitro requires an excess ofdsRNA relative to mRNA. In contrast, a few molecules of dsRNA per cellcan inhibit gene expression in vivo (Fire et al., Nature, 391: 806-811(1998); Kennerdell and Carthew, Cell, 95:1017-1026 (1998)). Thedifference between the stoichiometry of dsRNA to target mRNA in vivo andin vitro should not be surprising in that most in vitro reactions areless efficient than their corresponding in vivo processes.Interestringly, incubation of the dsRNA in the lysate greatlypotentiated its activity for RNAi, indicating that it is either modifiedor becomes associated with other factors or both. Perhaps a small numberof molecules is effective in inhibiting the targeted mRNA in vivobecause the injected dsRNA has been activated by a process similar tothat reported here for RNAi in Drosophila lysates.

Example 2 Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNAat 21 to 23

Nucleotide Intervals

Methods and Material

In vitro RNAi

In vitro RNAi reactions and lysate preparation were as described inExample 1 (Tuschl et al., Genes Dev., 13:3191-7 (1999)) except that thereaction contained 0.03 g/ml creatine kinase, 25 μM creatine phosphate(Fluka), and 1 mM ATP. Creatine phosphate was freshly dissolved at 500mM in water for each experiment. GTP was omitted from the reactions,except in FIGS. 2 and 3.

RNA Synthesis.

Pp-luc and Rr-luc mRNAs and Pp- and Rr-dsRNAs (including dsRNA ‘B’ inFIG. 6) were synthesized by in vitro transcription as describedpreviously (Tuschl et al., Genes Dev., 13:3191-7 (1999)). To generatetranscription templates for dsRNA ‘C’ the 5′ sense RNA primer wasgcgtaatacgactcactataGAACAAAGGAAACGGATGAT (SEQ ID NO: 2) and the 3′ senseRNA primer was GAAGAAGTTATTCTCCAAAA (SEQ ID NO: 3); the 5′ asRNA primerwas gcgtaatacgactcactataGAAGAAGTTATTCTCCAAAA (SEQ ID NO: 4) and the 3′asRNA primer was GAACAAAGGAAACGGATGAT (SEQ ID NO: 5). For dsRNA ‘A’ the5′ sense RNA primer was gcgtaatacgactcactataGTAGCGCGGTGTATTATACC (SEQ IDNO: 6) and the 3′ sense RNA primer was GTACAACGTCAGGTTTACCA (SEQ ID NO:7); the 5′ asRNA primer was gcgtaatacgactcactataGTACAACGTCAGGTTTACCA(SEQ ID NO: 8) and the 3′ asRNA primer was GTAGCGCGGTGTATTATACC (SEQ IDNO: 9) (lowercase, T7 promoter sequence).

mRNAs were 5′-end-labeled using guanylyl transferase (Gibco/BRL),S-adenosyl methionine (Sigma), and α-³²P-GTP (3000 Ci/mmol; New EnglandNuclear) according to the manufacturer's directions. Radiolabeled RNAswere purified by poly(A) selection using the Poly(A) Tract III kit(Promega). Nonradioactive 7-methyl-guanosine- and adenosine-capped RNAswere synthesized in in vitro transcription reactions with a 5-foldexcess of 7-methyl-G(5′)ppp(5′)G or A(5′)ppp(5′)G relative to GTP. Capanalogs were purchased from New England Biolabs.

ATP Depletion and Protein Synthesis Inhibition

ATP was depleted by incubating the lysate for 10 minutes at 25° C. with2 mM glucose and 0.1 U/ml hexokinase (Sigma). Protein synthesisinhibitors were purchased from Sigma and dissolved in absolute ethanolas 250-fold concentrated stocks. The final concentrations of inhibitorsin the reaction were: anisomycin, 53 mg/ml; cycloheximide, 100 mg/ml;chloramphenicol, 100 mg/ml. Relative protein synthesis was determined bymeasuring the activity of Rr luciferase protein produced by translationof the Rr-luc mRNA in the RNAi reaction after 1 hour as describedpreviously (Tuschl et al., Genes Dev., 13:3191-7 (1999)).

Analysis of dsRNA Processing

Internally α-³²P-ATP-labeled dsRNAs (505 bp Pp-luc or 501 Rr-luc) or7-methyl-guanosine-capped Rr-luc antisense RNA (501 nt) were incubatedat 5 nM final concentration in the presence or absence of unlabeledmRNAs in Drosophila lysate for 2 hours in standard conditions. Reactionswere stopped by the addition of 2× proteinase K buffer and deproteinizedas described previously (Tuschl et al., Genes Dev., 13:3191-3197(1999)). Products were analyzed by electrophoresis in 15% or 18%polyacrylamide sequencing gels. Length standards were generated bycomplete RNase Ti digestion of α-³²P-ATP-labeled 501 nt Rr-luc sense RNAand asRNA.

For analysis of mRNA cleavage, 5′-³²P-radiolabeled mRNA (describedabove) was incubated with dsRNA as described previously (Tuschl et al.,Genes Dev., 13:3191-3197 (1999)) and analyzed by electrophoresis in 5%(FIG. 5B) and 6% (FIG. 6C) polyacrylamide sequencing gels. Lengthstandards included commercially available RNA size standards (FMCBioproducts) radiolabeled with guanylyl transferase as described aboveand partial base hydrolysis and RNase Ti ladders generated from the5′-radiolabeled mRNA.

Deamination Assay

Internally α-³²P-ATP-labeled dsRNAs (5 nM) were incubated in Drosophilalysate for 2 hours at standard conditions. After deproteinization,samples were run on 12% sequencing gels to separate full-length dsRNAsfrom the 21-23 nt products. RNAs were eluted from the gel slices in 0.3M NaCl overnight, ethanol-precipitated, collected by centrifugation, andredissolved in 20 μA water. The RNA was hydrolyzed into nucleoside5-phosphates with nuclease P1 (10 μl reaction containing 8 μl RNA inwater, 30 mM KOAc pH 5.3, 10 mM ZnSO₄, 10 μg or 3 units nuclease P1, 3hours, 50° C.). Samples (1 ml) were co-spotted with non-radioactive5-mononucleotides [0.05 O.D. units (A₂₆₀) of pA, pC, pG, pI, and pU] oncellulose HPTLC plates (EM Merck) and separated in the first dimensionin isobutyric acid/25% ammonia/water (66/1/33, v/v/v) and in the seconddimension in 0.1M sodium phosphate, pH 6.8/ammonium sulfate/1-propanol(100/60/2, v/w/v; Silberklang et al., 1979). Migration of thenon-radioactive internal standards was determined by UV-shadowing.

Results and Discussion

RNAi Requires ATP

As described in Example 1, Drosophila embryo lysates faithfullyrecapitulate RNAi (Tuschl et al., Genes Dev., 13:3191-7 (1999)).Previously, dsRNA-mediated gene silencing was monitored by measuring thesynthesis of luciferase protein from the targeted mRNA. Thus, these RNAireactions contained an ATP-regenerating system, needed for the efficienttranslation of the mRNA. To test if ATP was, in fact, required for RNAi,the lysates were depleted for ATP by treatment with hexokinase andglucose, which converts ATP to ADP, and RNAi was monitored directly byfollowing the fate of ³²P-radiolabeled Renilla reniformis luciferase(Rr-luc) mRNA (FIG. 6). Treatment with hexokinase and glucose reducedthe endogenous ATP level in the lysate from 250 μM to below 10 μM. ATPregeneration required both exogenous creatine phosphate and creatinekinase, which acts to transfer a high-energy phosphate from creatinephosphate to ADP. When ATP-depleted extracts were supplemented witheither creatine phosphate or creatine kinase separately, no RNAi wasobserved. Therefore, RNAi requires ATP in vitro. When ATP, creatinephosphate, and creatine kinase were all added together to reactionscontaining the ATP-depleted lysate, dsRNA-dependent degradation of theRr-luc mRNA was restored (FIG. 6). The addition of exogenous ATP was notrequired for efficient RNAi in the depleted lysate, provided that bothcreatine phosphate and creatine kinase were present, demonstrating thatthe endogenous concentration (250 mM) of adenosine nucleotide issufficient to support RNAi. RNAi with a Photinus pyralis luciferase(Pp-luc) mRNA was also ATP-dependent.

The stability of the Rr-luc mRNA in the absence of Rr-dsRNA was reducedin ATP-depleted lysates relative to that observed when the energyregenerating system was included, but decay of the mRNA under theseconditions did not display the rapid decay kinetics characteristic ofRNAi in vitro, nor did it generate the stable mRNA cleavage productscharacteristic of dsRNA-directed RNAi. These experiments do notestablish if the ATP requirement for RNAi is direct, implicating ATP inone or more steps in the RNAi mechanism, or indirect, reflecting a rolefor ATP in maintaining high concentrations of another nucleosidetriphosphate in the lysate.

Translation is not Required for RNAi In Vitro

The requirement for ATP suggested that RNAi might be coupled to mRNAtranslation, a highly energy-dependent process. To test thispossibility, various inhibitors of protein synthesis were added to thereaction by preparing a denaturing agarose-gel analysis of5′-32P-radiolabeled Pp-luc mRNA after incubation for indicated times ina standard RNAi reaction with and without protein synthesis inhibitors.The eukaryotic translation inhibitors anisomycin, an inhibitor ofinitial peptide bond formation, cycloheximide, an inhibitor of peptidechain elongation, and puromycin, a tRNA mimic which causes prematuretermination of translation (Cundliffe, Antibiotic Inhibitors of RibosomeFunction. In The Molecular Basis of Antibiotic Action, E. Gale, E.Cundliffe, P. Reynolds, M. Richmond and M. Warning, eds. (New York:Wiley), pp. 402-547. (1981)) were tested. Each of these inhibitorsreduced protein synthesis in the Drosophila lysate by more than1,900-fold (FIG. 7A). In contrast, chloramphenicol, an inhibitor ofDrosophila mitochondrial protein synthesis (Page and Orr-Weaver, Dev.Biol., 183:195-207 (1997)), had no effect on translation in the lysates(FIG. 7A). Despite the presence of anisomycin, cycloheximide, orchloramphenicol, RNAi proceeded at normal efficiency. Puromycin also didnot perturb efficient RNAi. Thus, protein synthesis is not required forRNAi in vitro.

Translational initiation is an ATP-dependent process that involvesrecognition of the 7-methyl guanosine cap of the mRNA (Kozak, Gene,234:187-208 (1999); Merrick and Hershey, The Pathway and Mechanism ofEukaryotic Protein Synthesis. In Translational Control, J. Hershey, M.Mathews and N. Sonenberg, eds. (Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory Press), pp. 31-69 (1996)). The Drosophila lysate usedto support RNAi in vitro also recapitulates the cap-dependence oftranslation; Pp-luc mRNA with a 7-methyl-guanosine cap was translatedgreater than ten-fold more efficiently than was the same mRNA with anA(5′)ppp(5′)G cap (FIG. 7B). Both RNAs were equally stable in theDrosophila lysate, showing that this difference in efficiency cannot bemerely explained by more rapid decay of the mRNA with an adenosine cap(see also Gebauer et al., EMBO J., 18:6146-54 (1999)). Although thetranslational machinery can discriminate between Pp-luc mRNAs with7-methyl-guanosine and adenosine caps, the two mRNAs were equallysusceptible to RNAi in the presence of Pp-dsRNA (FIG. 7C). These resultssuggest that steps in cap recognition are not involved in RNAi.

dsRNA is Processed to 21-23 nt Species

RNAs 25 nt in length are generated from both the sense and anti-sensestrands of genes undergoing post-transcriptional gene silencing inplants (Hamilton and Baulcombe, Science, 286:950-2 (1999)). Denaturingacrylamide-gel analysis of the products formed in a two-hour incubationof uniformly ³²P-radiolabeled dsRNAs and capped asRNA in lysate understandard RNAi conditions, in the presence or absence of target mRNAs. Itwas found that dsRNA is also processed to small RNA fragments. Whenincubated in lysate, approximately 15% of the input radioactivity ofboth the 501 bp Rr-dsRNA and the 505 bp Pp-dsRNA appeared in 21 to 23 ntRNA fragments. Because the dsRNAs are more than 500 bp in length, the15% yield of fragments implies that multiple 21-23 nt RNAs are producedfrom each full-length dsRNA molecule. No other stable products weredetected. The small RNA species were produced from dsRNAs in which bothstrands were uniformly ³²P-radiolabeled. Formation of the 21-23 nt RNAsfrom the dsRNA did not require the presence of the corresponding mRNA,demonstrating that the small RNA species is generated by processing ofthe dsRNA, rather than as a product of dsRNA-targeted mRNA degradation.It was noted that 22 nucleotides corresponds to two turns of an A-formRNA-RNA helix.

When dsRNAs radiolabeled within either the sense or the anti-sensestrand were incubated with lysate in a standard RNAi reaction, 21-23 ntRNAs were generated with comparable efficiency. These data support theidea that the 21-23 nt RNAs are generated by symmetric processing of thedsRNA. A variety of data support the idea that the 21-23 nt RNA isefficiently generated only from dsRNA and is not the consequence of aninteraction between single-stranded RNA and the dsRNA. First, a³²P-radiolabeled 505 nt Pp-luc sense RNA or asRNA was not efficientlyconverted to the 21-23 nt product when it was incubated with 5 nMnonradioactive 505 bp Pp-dsRNA. Second, in the absence of mRNA, a 501 nt7-methyl-guanosine-capped Rr-asRNA produced only a barely detectableamount of 21-23 nt RNA (capped single-stranded RNAs are as stable in thelysate as dsRNA, Tuschl et al., Genes Dev., 13:3191-7(1999)), probablydue to a small amount of dsRNA contaminating the anti-sense preparation.However, when Rr-luc mRNA was included in the reaction with the³²P-radiolabeled, capped Rr-asRNA, a small amount of 21-23 nt productwas generated, corresponding to 4% of the amount of 21-23 nt RNAproduced from an equimolar amount of Rr-dsRNA. This result is unlikelyto reflect the presence of contaminating dsRNA in the Rr-asRNApreparation, since significantly more product was generated from theasRNA in the presence of the Rr-luc mRNA than in the absence. Instead,the data suggest that asRNA can interact with the complementary mRNAsequences to form dsRNA in the reaction and that the resulting dsRNA issubsequently processed to the small RNA species. Rr-asRNA can support alow level of bona fide RNAi in vitro (see below), consistent with thisexplanation.

It was next asked if production of the 21-23 nt RNAs from dsRNA requiredATP. When the 505 bp Pp-dsRNA was incubated in a lysate depleted for ATPby treatment with hexokinase and glucose, 21-23 nt RNA was produced,albeit 6 times slower than when ATP was regenerated in the depletedlysate by the inclusion of creatine kinase and creatine phosphate.Therefore, ATP may not be required for production of the 21-23 nt RNAspecies, but may instead simply enhance its formation. Alternatively,ATP may be required for processing of the dsRNA, but at a concentrationless than that remaining after hexokinase treatment. The molecular basisfor the slower mobility of the small RNA fragments generated in theATP-depleted lysate is not understood.

Wagner and Sun (Wagner and Sun, Nature, 391:744-745 (1998)) and Sharp(Sharp, Genes Dev., 13:139-41 (1999)) have speculated that therequirement for dsRNA in gene silencing by RNAi reflects the involvementof a dsRNA-specific adenosine deaminase in the process. dsRNA adenosinedeaminases unwind dsRNA by converting adenosine to inosine, which doesnot base-pair with uracil. dsRNA adenosine deaminases function in thepost-transcriptional editing of mRNA (for review see Bass, TrendsBiochem. Sci., 22:157-62 (1997)). To test for the involvement of dsRNAadenosine deaminase in RNAi, the degree of conversion of adenosine toinosine in the 501 bp Rr-luc and 505 bp Pp-luc dsRNAs after incubationwith Drosophila embryo lysate in a standard in vitro RNAi reaction wasexamined. Adenosine deamination in full-length dsRNA and the 21-23 ntRNA species was assessed by two-dimensional thin-layer chromatography.Inorganic phosphate (P_(i),) was produced by the degradation ofmononucleotides by phosphatases that contaminate commercially availablenuclease P1 (Auxilien et al., J. Mol. Biol., 262:437-458 (1996)). Thedegree of adenosine deamination in the 21-23 nt species was alsodetermined. The full-length dsRNA radiolabeled with [³²P]-adenosine wasincubated in the lysate, and both the full-length dsRNA and the 21-23 ntRNA products were purified from a denaturing acrylamide gel, cleaved tomononucleotides with nuclease P1, and analyzed by two-dimensionalthin-layer chromatography.

A significant fraction of the adenosines in the full-length dsRNA wereconverted to inosine after 2 hours (3.1% and 5.6% conversion for Pp-lucand Rr-luc dsRNAs, respectively). In contrast, only 0.4% (Pp-dsRNA) or0.7% (Rr-dsRNA) of the adenosines in the 21-23 nt species weredeaminated. These data imply that fewer than 1 in 27 molecules of the21-23 nt RNA species contain an inosine. Therefore, it is unlikely thatdsRNA-dependent adenosine deamination within the 21-23 nt species isrequired for its production. asRNA Generates a Small Amount of RNAi invitro When mRNA was ³²P-radiolabeled within the 5′-7-methyl-guanosinecap, stable 5′ decay products accumulated during the RNAi reaction. Suchstable 5′ decay products were observed for both the Pp-luc and Rr-lucmRNAs when they were incubated with their cognate dsRNAs. Previously, itwas reported that efficient RNAi does not occur when asRNA is used inplace of dsRNA (Tuschl et al., Genes Dev., 13:3191-7 (1999)).Nevertheless, mRNA was measurably less stable when incubated with asRNAthan with buffer (FIGS. 8A and 8B). This was particularly evident forthe Rr-luc mRNA: approximately 90% of the RNA remained intact after a3-hour incubation in lysate, but only 50% when asRNA was added. Lessthan 5% remained when dsRNA was added. Interestingly, the decrease inmRNA stability caused by asRNA was accompanied by the formation of asmall amount of the stable 5′-decay products characteristic of the RNAireaction with dsRNA. This finding parallels the observation that a smallamount of 21-23 nt product formed from the asRNA when it was incubatedwith the mRNA (see above) and lends strength to the idea that asRNA canenter the RNAi pathway, albeit inefficiently.

mRNA Cleavage Sites are Determined by the Sequence of the dsRNA

The sites of mRNA cleavage were examined using three different dsRNAs,‘A,’ ‘B,’ and ‘C,’ displaced along the Rr-luc sequence by approximately100 nts. Denaturing acrylamide-gel analysis of the stable, 5′-cleavageproducts produced after incubation of the Rr-luc mRNA for the indicatedtimes with each of the three dsRNAs, ‘A,’ ‘B,’ and ‘C,’ or with buffer(.O slashed.) was performed. The positions of these relative to theRr-luc mRNA sequence are shown in FIG. 9. Each of the three dsRNAs wasincubated in a standard RNAi reaction with Rr-luc mRNA ³²P-radiolabeledwithin the 5′-cap. In the absence of dsRNA, no stable 5′-cleavageproducts were detected for the mRNA, even after 3 hours of incubation inlysate. In contrast, after a 20-minute incubation, each of the threedsRNAs produced a ladder of bands corresponding to a set of mRNAcleavage products characteristic for that particular dsRNA. For eachdsRNA, the stable, 5′ mRNA cleavage products were restricted to theregion of the Rr-luc mRNA that corresponded to the dsRNA (FIGS. 9 and10). For dsRNA ‘A,’ the lengths of the 5′ cleavage products ranged from236 to just under ˜750 nt; dsRNA ‘A’ spans nucleotides 233 to 729 of theRr-luc mRNA. Incubation of the mRNA with dsRNA ‘B’ produced mRNA5′-cleavage products ranging in length from 150 to ˜600 nt; dsRNA ‘B’spans nucleotides 143 to 644 of the mRNA. Finally, dsRNA ‘C’ producedmRNA cleavage products from 66 to ˜500 nt in length. This dsRNA spansnucleotides 50 to 569 of the Rr-luc mRNA. Therefore, the dsRNA not onlyprovides specificity for the RNAi reaction, selecting which mRNA fromthe total cellular mRNA pool will be degraded, but also determines theprecise positions of cleavage along the mRNA sequence.

The mRNA is Cleaved at 21-23 Nucleotide Intervals

To gain further insight into the mechanism of RNAi, the positions ofseveral mRNA cleavage sites for each of the three dsRNAs were mapped(FIG. 10). High resolution denaturing acrylamide-gel analysis of asubset of the 5′-cleavage products described above was performed.Remarkably, most of the cleavages occurred at 21-23 nt intervals (FIG.10). This spacing is especially striking in light of our observationthat the dsRNA is processed to a 21-23 nt RNA species and the finding ofHamilton and Baulcombe that a 25 nt RNA correlates withpost-transcriptional gene silencing in plants (Hamilton and Baulcombe,Science, 286:950-2 (1999)). Of the 16 cleavage sites we mapped (2 fordsRNA ‘A,’ 5 for dsRNA ‘B,’ and 9 for dsRNA ‘C’), all but two reflectthe 21-23 nt interval. One of the two exceptional cleavages was a weakcleavage site produced by dsRNA ‘C’ (indicated by an open blue circle inFIG. 10). This cleavage occurred 32 nt 5′ to the next cleavage site. Theother exception is particularly intriguing. After four cleavages spaced21-23 nt apart, dsRNA ‘C’ caused cleavage of the mRNA just nine nt 3′ tothe previous cleavage site (red arrowhead in FIG. 10). This cleavageoccurred in a run of seven uracil residues and appears to “reset” theruler for cleavage; the next cleavage site was 21-23 nt 3′ to theexceptional site. The three subsequent cleavage sites that we mappedwere also spaced 21-23 nt apart. Curiously, of the sixteen cleavagesites caused by the three different dsRNAs, fourteen occur at uracilresidues. The significance of this finding is not understood, but itsuggests that mRNA cleavage is determined by a process which measures21-23 nt intervals and which has a sequence preference for cleavage aturacil. Results show that the 21-23 nt RNA species produced byincubation of 500 bp dsRNA in the lysate caused sequence-specificinterference in vitro when isolated from an acrylamide gel and added toa new RNAi reaction in place of the full-length dsRNA.

A Model for dsRNA-Directed mRNA Cleavage

Without wishing to be bound by theory, the biochemical data describedherein, together with recent genetic experiments in C. elegans andNeurospora (Cogoni and Macino, Nature, 399:166-9 (1999); Grishok et al.,Science, 287: 2494-7 (2000); Ketting et al., Cell, 99:133-41 (1999);Tabara et al., Cell, 99:123-32 (1999)), suggest a model for how dsRNAtargets mRNA for destruction (FIG. 11). In this model, the dsRNA isfirst cleaved to 21-23 nt long fragments in a process likely to involvegenes such as the C. elegans loci rde-1 and rde-4. The resultingfragments, probably as short asRNAs bound by RNAi-specific proteins,would then pair with the mRNA and recruit a nuclease that cleaves themRNA. Alternatively, strand exchange could occur in a protein-RNAcomplex that transiently holds a 21-23 nt dsRNA fragment close to themRNA. Separation of the two strands of the dsRNA following fragmentationmight be assisted by an ATP-dependent RNA helicase, explaining theobserved ATP enhancement of 21-23 nt RNA production.

It is likely that each small RNA fragment produces one, or at most two,cleavages in the mRNA, perhaps at the 5′ or 3′ ends of the 21-23 ntfragment. The small RNAs may be amplified by an RNA-directed RNApolymerase such as that encoded by the ego-1 gene in C. elegans (Smardonet al., Current Biology, 10:169-178 (2000)) or the qde-1 gene inNeurospora (Cogoni and Macino, Nature, 399:166-9 (1999)), producinglong-lasting post-transcriptional gene silencing in the absence of thedsRNA that initiated the RNAi effect. Heritable RNAi in C. elegansrequires the rde-1 and rde-4 genes to initiate, but not to persist insubsequent generations. The rde-2, rde-3, and mut-7 genes in C. elegansare required in the tissue where RNAi occurs, but are not required forinitiation of heritable RNAi (Grishok et al., Science, in press 2000).These ‘effector’ genes (Grishok et al., Science, in press 2000) arelikely to encode proteins functioning in the actual selection of mRNAtargets and in their subsequent cleavage. ATP may be required at any ofa number of steps during RNAi, including complex formation on the dsRNA,strand dissociation during or after dsRNA cleavage, pairing of the 21-23nt RNAs with the target mRNA, mRNA cleavage, and recycling of thetargeting complex. Testing these ideas with the in vitro RNAi systemwill be an important challenge for the future. Some genes involved inRNAi are also important for transposon silencing and co-suppresion.Co-suppression is a broad biological phenomenon spanning plants, insectsand perhaps humans. The most likely mechanism in Drosophila melanogasteris transcriptional silencing (Pal-Bhanra et al, Cell 99: 35-36. Thus,21-23 nt fragments are likely to be involved in transcriptional control,as well as in post-transcriptional cotrol

Example 3 Isolated 21-23 Mers Caused Sequence-Specific Interference whenAdded to a New RNAi Reaction

Isolation of 21-23 Nt Fragments from Incubation Reaction of 500 Bp dsRNAin Lysate.

Double-stranded RNA (500 bp from) was incubated at 10 nM concentrationin Drosophila embryo lysate for 3 h at 25° C. under standard conditionsas described herein. After deproteinization of the sample, the 21-23 ntreaction products were separated from to unprocessed dsRNA by denaturingpolyacrylamide (15%) gel electrophoresis. For detection of thenon-radiolabeled 21-23 nt fragments, an incubation reaction withradiolabeled dsRNA was loaded in a separate lane of the same gel. Gelslices containing the non-radioactive 21-23 nt fragments were cut outand the 21-23 nt fragments were eluted from the gel slices at 4° C.overnight in 0.4 ml 0.3 M NaCl. The RNA was recovered from thesupernatant by ethanol precipitation and centrifugation. The RNA pelletwas dissolved in 10 μl of lysis buffer. As control, gel slices slightlyabove and below the 21-23 nt band were also cut out and subjected to thesame elution and precipitation procedures. Also, a non-incubated dsRNAloaded on the 15% gel and a gel slice corresponding to 21-23 ntfragments was cut out and eluted. All pellets from the controlexperiments were dissolved in 10 μl lysis buffer. The losses of RNAduring recovery from gel slices by elution are approx. 50%.

Incubation of Purified 21-23 Nt Fragments in a Translation-Based RNAiAssay

1 μl of the eluted 21-23 mer or control RNA solution was used for astandard 10 μl RNAi incubation reaction (see above). The 21-23 mers werepreincubated in the lysate containing reaction mixture for 10 or 30 minbefore the addition of the target and control mRNA. Duringpre-incubation, proteins involved in RNA interference may re-associatewith the 21-23 mers due to a specific signal present on these RNAs. Theincubation was continued for another hour to allow translation of thetarget and control mRNAs. The reaction was quenched by the addition ofpassive lysis buffer (Promega), and luciferase activity was measured.The RNA interference is the expressed as the ratio of target to controlluciferase activity normalized by an RNA-free buffer control. Specificsuppression of the target gene was observed with either 10 or 30 minutespreincubation. The suppression was reproducible and reduced the relativeratio of target to control by 2-3 fold. None of the RNA fragmentsisolated as controls showed specific interference. For comparison,incubation of 5 nM 500 bp dsRNA (10 min pre-incubation) affects therelative ratio of control to target gene approx. 30-fold.

Stability of Isolated 21-23 Nt Fragments in a New Lysate IncubationReaction.

Consistent with the observation of RNAi mediated by purified 21-23 ntRNA fragment, it was found that 35% of the input 21-23 nt RNA persistsfor more than 3 h in such an incubation reaction. This suggests thatcellular factors associate with the deproteinized 21-23 nt fragments andreconstitute a functional mRNA-degrading particle. Signals connectedwith these 21-23 nt fragments, or their possible double stranded natureor specific lengths are likely responsible for this observation. The21-23 nt fragments have a terminal 3′ hydroxyl group, as evidenced byaltered mobility on a sequencing gel following periodate treatment andbeta-elimination.

Example 4 21-23-Mers Purified by Non-Denaturing Methods CausedSequence-Specific Interference when Added to a New RNAi Reaction

Fifty nanomolar double-stranded RNA (501 bp Rr-luc dsRNA, as describedin example 1) was incubated in a 1 ml in vitro reaction with lysate at25° C. (see example 1). The reaction was then stopped by the addition ofan equal volume of 2×PK buffer (see example 1) and proteinase K wasadded to a final concentration of 1.8 μg/μl. The reaction was incubatedfor an additional 1 h at 25° C., phenol extracted, and then the RNAswere precipitated with 3 volumes of ethanol. The ethanol precipitate wascollected by centrifugation, and the pellet was resuspended in 100 μl oflysis buffer and applied to a Superdex HR 200 10/30 gel filtrationcolumn (Pharmacia) run in lysis buffer at 0.75 ml/min. 200 μl fractionswere collected from the column. Twenty μl of 3 M sodium acetate and 20μg glycogen was added to each fraction, and the RNA was recovered byprecipitation with 3 volumes of ethanol. The precipitates wereresuspended in 30 μl of lysis buffer. Column profiles following thefractionation of 32P-labeled input RNA are shown in FIG. 13A.

One microliter of each resuspended fraction was tested in a 10 μlstandard in vitro RNAi reaction (see example 1). This procedure yields aconcentration of RNA in the in vitro RNAi reaction that is approximatelyequal to the concentration of that RNA species in the original reactionprior to loading on the column. The fractions were preincubated in thelysate containing reaction mixture for 30 min before the addition of 10nM Rr-luc mRNA target and 10 nM Pp-luc control mRNA. Duringpre-incubation, proteins involved in RNA interference may re-associatewith the 21-23-mers due to a specific signal present on these RNAs. Theincubation was continued for another three hours to allow translation ofthe target and control mRNAs. The reaction was quenched by the additionof passive lysis buffer (Promega), and luciferase activity was measured.The suppression of Rr-luc mRNA target expression by the purified 21-23nt fragments was reproducible and reduced the relative ratio of targetto control by >30-fold, an amount comparable to a 50 nM 500 bp dsRNAcontrol. Suppression of target mRNA expression was specific: little orno effect on the expression of the Pp-luc mRNA control was observed.

The data show that the both the fractions containing uncleaved dsRNA(fractions 3-5) or long, partially cleaved dsRNA (fractions 7-13) andthe fractions containing the fully processed 21-23 nt siRNAs (fractions41-50) mediate effective RNA interference in vitro (FIG. 13B).Suppression of target mRNA expression was specific: little or no effecton the expression of the Pp-luc mRNA control was observed (FIG. 13C).These data, together with those in the earlier examples, demonstratethat the 21-23 nt siRNAs are (1) true intermediates in the RNAi pathwayand (2) effective mediators of RNA interference in vitro.

Example 5 21-Nucleotide siRNA Duplexes Mediate RNA Interference in HumanTissue Cultures

Methods

RNA Preparation

21 nt RNAs were chemically synthesized using Expedite RNAphosphoramidites and thymidine phosphoramidite (Proligo, Germany).Synthetic oligonucleotides were deprotected and gel-purified (Elbashir,S. M., Lendeckel, W. & Tuschl, T., Genes & Dev. 15, 188-200 (2001)),followed by Sep-Pak C18 cartridge (Waters, Milford, Mass., USA)purification (Tuschl, t., et al., Biochemistry, 32:11658-11668 (1993)).The siRNA sequences targeting GL2 (Acc. X65324) and GL3 luciferase (Acc.U47296) corresponded to the coding regions 153-173 relative to the firstnucleotide of the start codon, siRNAs targeting RL (Acc. AF025846)corresponded to region 119-129 after the start codon. Longer RNAs weretranscribed with T7 RNA polymerase from PCR products, followed by geland Sep-Pak purification. The 49 and 484 bp GL2 or GL3 dsRNAscorresponded to position 113-161 and 113-596, respectively, relative tothe start of translation; the 50 and 501 bp RL dsRNAs corresponded toposition 118-167 and 118-618, respectively. PCR templates for dsRNAsynthesis targeting humanized GFP (hG) were amplified from pAD3(Kehlenbach, R. H., et al., J. Cell Biol., 141:863-874 (1998)), whereby50 and 501 bp hG dsRNA corresponded to position 118-167 and 118-618,respectively, to the start codon.

For annealing of siRNAs, 20 μM single strands were incubated inannealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2mM magnesium acetate) for 1 min at 90° C. followed by 1 h at 37° C. The37° C. incubation step was extended overnight for the 50 and 500 bpdsRNAs, and these annealing reactions were performed at 8.4 μM and 0.84μM strand concentrations, respectively.

Cell Culture

S2 cells were propagated in Schneider's Drosophila medium (LifeTechnologies) supplemented with 10% FBS, 100 units/ml penicillin, and100 μg/ml streptomycin at 25° C. 293, NIH/3T3, HeLa S3, COS-7 cells weregrown at 37° C. in Dulbecco's modified Eagle's medium supplemented with10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells wereregularly passaged to maintain exponential growth. 24 h beforetransfection at approx. 80% confluency, mammalian cells were trypsinizedand diluted 1:5 with fresh medium without antibiotics (1-3×10⁵ cells/ml)and transferred to 24-well plates (500 μl/well). S2 cells were nottrypsinized before splitting. Transfection was carried out withLipofectamine 2000 reagent (Life Technologies) as described by themanufacturer for adherent cell lines. Per well, 1.0 μg pGL2-Control(Promega) or pGL3-Control (Promega), 0.1 μg pRL-TK (Promega), and 0.28μg siRNA duplex or dsRNA, formulated into liposomes, were applied; thefinal volume was 600 μl per well. Cells were incubated 20 h aftertransfection and appeared healthy thereafter. Luciferase expression wassubsequently monitored with the Dual luciferase assay (Promega).Transfection efficiencies were determined by fluorescence microscopy formammalian cell lines after co-transfection of 1.1 μg hGFP-encodingpAD3²² and 0.28 μg invGL2 siRNA, and were 70-90%. Reporter plasmids wereamplified in XL-1 Blue (Strategene) and purified using the QiagenEndoFree Maxi Plasmid Kit.

Results

RNA interference (RNAi) is the process of sequence-specific,post-transcriptional gene silencing in animals and plants, initiated bydouble-stranded RNA (dsRNA) homologous in sequence to the silenced gene(Fire, A., Trends Genet., 15:358-363 (1999); Sharp, P. A. & Zamore, P.D., Science, 287:2431-2433 (2000); Sijen, T. & Kooter, J. M., Bioessays,22:520-531 (2000); Bass, B. L., Cell, 101:235-238 (2000); Hammond, S.M., et al., Nat. Rev. Genet., 2:110-119 (2001)). The mediators ofsequence-specific mRNA degradation are 21 and 22 nt small interferingRNAs (siRNAs) generated by RNase III cleavage from longer dsRNAs⁶⁻¹⁰(Hamilton, A. J. & Baulcombe, D. C, Science, 286:950-952 (1999);Hammond, S. M., et al., Nature, 404:293-296 (2000); Zamore, P. D., etal., Cell, 101:25-33 (2000); Bernstein, E., et al, Naature, 409:363-366(2001); Elbashir, S. M., et al., Genes & Dev., 15:188-200 (2001)). Asshown herein, 21 nt siRNA duplexes are able to specifically suppressreporter gene expression in multiple mammalian tissue cultures,including human embryonic kidney (293) and HeLa cells. In contrast to 50or 500 bp dsRNAs, siRNAs do not activate the interferon response. Theseresults indicate that siRNA duplexes are a general tool forsequence-specific inactivation of gene function in mammalian cells.

Base-paired 21 and 22 nt siRNAs with overhanging 3′ ends mediateefficient sequence-specific mRNA degradation in lysates prepared from D.melanogaster embryos (Elbashir, S. M., et al., Genes & Dev., 15:188-200(2001)). To test whether siRNAs are also capable of mediating RNAi intissue culture, 21 nt siRNA duplexes with symmetric 2 nt 3′ overhangsdirected against reporter genes coding for sea pansy (Renillareniformis) and two sequence variants of firefly (Photinus pyralis, GL2and GL3) luciferases (FIGS. 14A, 14B) were constructed. The siRNAduplexes were co-transfected with the reporter plasmid combinationspGL2/pRL or pGL3/pRL, into D. melanogaster Schneider S2 cells ormammalian cells using cationic liposomes. Luciferase activities weredetermined 20 h after transfection. In all cell lines tested, specificreduction of the expression of the reporter genes in the presence ofcognate siRNA duplexes was observed (FIGS. 15A-15J). Remarkably, theabsolute luciferase expression levels were unaffected by non-cognatesiRNAs, indicating the absence of harmful side effects by 21 nt RNAduplexes (e.g. FIGS. 16A-16D, for HeLa cells). In D. melanogaster S2cells (FIGS. 15A, 15B), the specific inhibition of luciferases wascomplete, and similar to results previously obtained for longer dsRNAs(Hammond, S. M., et al., Nature, 404:293-296 (2000); Caplen, N. J., etal., sGene, 252:95-105 (2000); Clemens, M & Williams, B., Cell,13:565-572 (1978); Ui-Tei, K., et al., FEBS Letters, 479:79-82 (2000)).In mammalian cells, where the reporter genes were 50- to 100-foldstronger expressed, the specific suppression was less complete (FIGS.15C-15J). GL2 expression was reduced 3- to 12-fold, GL3 expression 9- to25-fold, and RL expression 1- to 3-fold, in response to the cognatesiRNAs. For 293 cells, targeting of RL luciferase by RL siRNAs wasineffective, although GL2 and GL3 targets responded specifically (FIGS.15I, 15J). It is likely that the lack of reduction of RL expression in293 cells is due to its 5- to 20-fold higher expression compared to anyother mammalian cell line tested and/or to limited accessibility of thetarget sequence due to RNA secondary structure or associated proteins.Nevertheless, specific targeting of GL2 and GL3 luciferase by thecognate siRNA duplexes indicated that RNAi is also functioning in 293cells.

The 2 nt 3′ overhang in all siRNA duplexes, except for uGL2, wascomposed of (2′-deoxy) thymidine. Substitution of uridine by thymidinein the 3′ overhang was well tolerated in the D. melanogaster in vitrosystem, and the sequence of the overhang was uncritical for targetrecognition (Elbashir, S. M., et al., Genes & Dev., 15:188-200 (2001)).The thymidine overhang was chosen, because it is supposed to enhancenuclease resistance of siRNAs in the tissue culture medium and withintransfected cells. Indeed, the thymidine-modified GL2 siRNA was slightlymore potent than the unmodified uGL2 siRNA in all cell lines tested(FIGS. 15A, 15C, 15E, 15G, 15I). It is conceivable that furthermodifications of the 3′ overhanging nucleotides will provide additionalbenefits to the delivery and stability of siRNA duplexes.

In co-transfection experiments, 25 nM siRNA duplexes with respect to thefinal volume of tissue culture medium were used (FIGS. 15A-15J,16A-16F). Increasing the siRNA concentration to 100 nM did not enhancethe specific silencing effects, but started to affect transfectionefficiencies due to competition for liposome encapsulation betweenplasmid DNA and siRNA. Decreasing the siRNA concentration to 1.5 nM didnot reduce the specific silencing effect, even though the siRNAs werenow only 2- to 20-fold more concentrated than the DNA plasmids. Thisindicates that siRNAs are extraordinarily powerful reagents formediating gene silencing, and that siRNAs are effective atconcentrations that are several orders of magnitude below theconcentrations applied in conventional antisense or ribozyme genetargeting experiments.

In order to monitor the effect of longer dsRNAs on mammalian cells, 50and 500 bp dsRNAs cognate to the reporter genes were prepared. Asnon-specific control, dsRNAs from humanized GFP (hG) (Kehlenbach, R. H.,et al., J. Cell Biol., 141:863874 (1998)) was used. When dsRNAs wereco-transfected, in identical amounts (not concentrations) to the siRNAduplexes, the reporter gene expression was strongly and unspecificallyreduced. This effect is illustrated for HeLa cells as a representativeexample (FIGS. 16A-16D). The absolute luciferase activities weredecreased unspecifically 10- to 20-fold by 50 bp dsRNA, and 20- to200-fold by 500 bp dsRNA co-transfection, respectively. Similarunspecific effects were observed for COS-7 and NIH/3T3 cells. For 293cells, a 10- to 20-fold unspecific reduction was observed only for 500bp dsRNAs. Unspecific reduction in reporter gene expression by dsRNA >30bp was expected as part of the interferon response (Matthews, M.,Interactions between viruses and the cellular machinery for proteinsynthesis in Translational Control (eds., Hershey, J., Matthews, M. &Sonenberg, N.) 505-548 (Cold Spring Harbor Laboratory Press, Plainview,N. Y.; 1996); Kumar, M. & Carmichael, G. G., Microbiol. Mol. Biol. Rev.,62:1415-1434 (1998); Stark, G. R., et al., Annu. Rev. Biochem.,67:227-264 (1998)). Surprisingly, despite the strong unspecific decreasein reporter gene expression, additional sequence-specific,dsRNA-mediated silencing were reproducibly detected. The specificsilencing effects, however, were only apparent when the relativereporter gene activities were normalized to the hG dsRNA controls (FIGS.16E, 16F). A 2- to 10-fold specific reduction in response to cognatedsRNA was observed, also in the other three mammalian cell lines tested.Specific silencing effects with dsRNAs (356-1662 bp) were previouslyreported in CHO-K1 cells, but the amounts of dsRNA required to detect a2- to 4-fold specific reduction were about 20-fold higher than in ourexperiments (Ui-Tei, K., et al., FEBS Letters, 479:79-82 (2000)). Also,CHO-K1 cells appear to be deficient in the interferon response. Inanother report, 293, NIH/3T3, and BHK-21 cells were tested for RNAiusing luciferase/lacZ reporter combinations and 829 bp specific lacZ or717 bp unspecific GFP dsRNA (Caplen, N. J., et al., Gene, 252:95105(2000)). The failure of detecting RNAi in this case is likely due to theless sensitive luciferase/lacZ reporter assay and the length differencesof target and control dsRNA. Taken together, the results describedherein indicate that RNAi is active in mammalian cells, but that thesilencing effect is difficult to detect if the interferon system isactivated by dsRNA >30 bp.

The mechanism of the 21 nt siRNA-mediated interference process inmammalian cells remains to be uncovered, and silencing may occurpost-transcriptional and/or transcriptional. In D. melanogaster lysate,siRNA duplexes mediate post-transcriptional gene silencing byreconstitution of a siRNA-protein complexes (siRNPs), which are guidingmRNA recognition and targeted cleavage (Hammond, S. M., et al., Nature,404:293-296 (2000); Zamore, P. D., et al., Cell, 101:25-33 (2000);Elbashir, S. M., et al., Genes & Dev., 15:188-200 (2001)). In plants,dsRNA-mediated post-transcriptional silencing has also been linked toRNA-directed DNA methylation, which may also be directed by 21 nt siRNAs(Wassenegger, M., Plant Mol. Biol, 43:203-220 (2000); Finnegan, E. J.,et al., Curr. Biol, 11:R99-R102 (2000)). Methylation of promoter regionscan lead to transcriptional silencing (Metter, M. F., et al., EMBO J.,19:5194-5201 (2000)), but methylation in coding sequences must not(Wang, M.-B., RNA, 7:16-28 (2001)). DNA methylation and transcriptionalsilencing in mammals are well-documented processes (Kass, S. U., et al.,Trends Genet., 13:444-449 (1997); Razin, A., EMBO J, 17:4905-4908(1998)), yet they have not been linked to post-transcriptionalsilencing. Methylation in mammals is predominantly directed towards CpGresidues. Because there is no CpG in the RL siRNA, but RL siRNA mediatesspecific silencing in mammalian tissue culture, it is unlikely that DNAmethylation is critical for our observed silencing process. In summary,described herein, is siRNA-mediated gene silencing in mammalian cells.The use of 21 nt siRNAs holds great promise for inactivation of genefunction in human tissue culture and the development of gene-specifictherapeutics.

While this invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims

What is claimed is:
 1. A method of producing a double-stranded RNAmolecule of from 21 to 23 nucleotides in length that mediates RNAinterference of an mRNA, comprising: (a) chemically synthesizing thedouble-stranded RNA molecule in the form of two separate complementaryRNA strands which are not covalently linked and each RNA strand has alength of from 21 to 23 nucleotides, wherein the sequence of one strandhas sufficient sequence correspondence to the mRNA to mediate RNAinterference by directing cleavage of the mRNA within the region ofsequence correspondence, and (b) combining the strands of (a) underconditions under which the double-stranded RNA molecule is produced,thereby producing the double-stranded RNA molecule of from 21 to 23nucleotides that mediates RNA interference of the mRNA.
 2. The methodaccording to claim 1, wherein the RNA strands are recombinantlyproduced.
 3. The method of claim 1, wherein the mRNA is a mammaliancellular mRNA.
 4. The method of claim 1, wherein the RNA moleculecomprises a terminal 3′ hydroxyl group.
 5. The method of claim 1,wherein one or more nucleotides of the double-stranded RNA molecule area non-naturally occurring nucleotide.
 6. The method of claim 1, whereinone or more nucleotides of the double-stranded RNA molecule are adeoxyribonucleotide.
 7. The method of claim 1, wherein one or morenucleotides of the double-stranded RNA molecule are a non-standardnucleotide.
 8. The method of claim 1, wherein the mRNA is a human mRNA.9. The method of claim 1, wherein the mRNA is a viral mRNA.
 10. Themethod of claim 1, wherein the double-stranded RNA molecule is perfectlycomplementary to the mRNA.
 11. The method of claim 1, wherein thedouble-stranded RNA molecule is 21 nucleotides in length.
 12. The methodof claim 1, wherein the double-stranded RNA molecule is 22 nucleotidesin length.
 13. The method of claim 1, wherein the double-stranded RNAmolecule is 23 nucleotides in length.
 14. A method of producing adouble-stranded RNA molecule of from 21 to 23 nucleotides in length thatmediates RNA interference of an mRNA, comprising: chemicallysynthesizing the double-stranded RNA molecule such that the RNA is inthe form of two separate complementary RNA strands which are notcovalently linked and each RNA strand has a length of from 21 to 23nucleotides, wherein the sequence of one strand has sufficient sequencecorrespondence to the mRNA to mediate RNA interference of the mRNA, andthereby producing the double-stranded RNA molecule of from 21 to 23nucleotides that mediates RNA interference of the mRNA.
 15. The methodof claim 1, wherein the mRNA encodes a protein whose presence in a humanis associated with a disease or undesirable condition.
 16. The method ofclaim 1, wherein the double-stranded RNA molecule is effective to reduceexpression of the mRNA.
 17. The method of claim 1, further comprisingtesting the double-stranded RNA molecule to determine the level of RNAinterference effected by the double-stranded RNA.
 18. The method ofclaim 1, wherein each RNA strand of the double-stranded RNA moleculecomprises a terminal 3′ hydroxyl group.
 19. The method of claim 1,wherein the double-stranded RNA molecule comprises one or more naturallyoccurring nucleotides.
 20. The method of claim 1, wherein thedouble-stranded RNA molecule comprises one or more naturally occurringnucleotides and one or more non-naturally occurring nucleotides.
 21. Themethod of claim 14, wherein the double-stranded RNA molecule comprises aterminal 3′ hydroxyl group.
 22. The method of claim 14, wherein each RNAstrand of the double-stranded RNA molecule comprises a terminal 3′hydroxyl group.
 23. The method of claim 14, wherein the double-strandedRNA molecule comprises one or more non-naturally occurring nucleotides.24. The method of claim 14, wherein the double-stranded RNA moleculecomprises one or more non-standard nucleotides.
 25. The method of claim14, wherein the double-stranded RNA molecule comprises one or moredeoxyribonucleotides.
 26. The method of claim 14, wherein thedouble-stranded RNA molecule comprises one or more naturally occurringnucleotides.
 27. The method of claim 14, wherein the double-stranded RNAmolecule comprises one or more naturally occurring nucleotides and oneor more non-naturally occurring nucleotides.
 28. The method of claim 14,wherein the mRNA is a mammalian cellular mRNA.
 29. The method of claim14, wherein the mRNA is a human mRNA.
 30. The method of claim 14,wherein the mRNA encodes a protein whose presence in a human isassociated with a disease or undesirable condition.
 31. The method ofclaim 14, wherein the mRNA is a viral mRNA.
 32. The method of claim 14,wherein the double-stranded RNA molecule is perfectly complementary tothe mRNA.
 33. The method of claim 14, wherein a strand of thedouble-stranded RNA molecule is 21 nucleotides in length.
 34. The methodof claim 14, wherein a strand of the double-stranded RNA molecule is 22nucleotides in length.
 35. The method of claim 14, wherein a strand ofthe double-stranded RNA molecule is 23 nucleotides in length.
 36. Amethod of producing a double-stranded RNA molecule of 21 to 23nucleotides in length that mediates RNA interference, comprising:synthesizing the double-stranded RNA molecule such that the RNA is inthe form of two separate complementary RNA strands which are notcovalently linked and each RNA strand has a length of from 21 to 23nucleotides, wherein the sequence of one strand has sufficient sequencecomplementarity to a target mRNA to mediate RNA interference of thetarget mRNA, and wherein the double-stranded RNA molecule comprises oneor more of: a non-naturally occurring nucleotide, a non-standardnucleotide, or a deoxyribonucleotide, thereby producing thedouble-stranded RNA molecule.
 37. The method of claim 36, wherein astrand of the double-stranded RNA molecule is 21 nucleotides in length.38. The method of claim 36, wherein a strand of the double-stranded RNAmolecule is 22 nucleotides in length.
 39. The method of claim 36,wherein a strand of the double-stranded RNA molecule is 23 nucleotidesin length.
 40. The method of claim 16, wherein the expression of themRNA is determined using an in vitro RNA interference assay.
 41. Amethod of producing a double-stranded RNA molecule of 21 to 23nucleotides in length that mediates RNA interference, comprising:synthesizing the double-stranded RNA molecule such that the RNA is inthe form of two separate complementary RNA strands which are notcovalently linked and each RNA strand has a length of from 21 to 23nucleotides, wherein one strand of the double-stranded RNA molecule isperfectly complementary to a target mRNA to mediate RNA interference ofthe target mRNA, thereby producing the double-stranded RNA molecule. 42.The method of claim 41, wherein a strand of the double-stranded RNAmolecule is 21 nucleotides in length.
 43. The method of claim 41,wherein a strand of the double-stranded RNA molecule is 22 nucleotidesin length.
 44. The method of claim 41, wherein a strand of thedouble-stranded RNA molecule is 23 nucleotides in length.
 45. The methodof claim 41, wherein the expression of the mRNA is determined using anin vitro RNA interference assay.
 46. The method of claim 41, whereinboth strands of the double-stranded RNA molecule are 21 nucleotides inlength.
 47. The method of claim 41, wherein both strands of thedouble-stranded RNA molecule are 22 nucleotides in length.
 48. Themethod of claim 41, wherein both strands of the double-stranded RNAmolecule are 23 nucleotides in length.
 49. The method of claim 41,wherein the double-stranded RNA molecule comprises one or morenon-naturally occurring nucleotides.
 50. The method of claim 41, whereinthe double-stranded RNA molecule comprises one or more non-standardnucleotides.
 51. The method of claim 41, wherein the double-stranded RNAmolecule comprises one or more deoxyribonucleotides.
 52. The method ofclaim 41, wherein the mRNA is a cellular mRNA.
 53. The method of claim41, wherein the mRNA is an oncogene mRNA.
 54. The method of claim 41,wherein the mRNA encodes a viral protein.
 55. The method of claim 1,wherein the mRNA is a cellular mRNA.
 56. The method of claim 1, whereinthe mRNA is an oncogene mRNA.
 57. The method of claim 1, wherein themRNA encodes a viral protein.
 58. The method of claim 14, wherein themRNA is a cellular mRNA.
 59. The method of claim 14, wherein the mRNA isan oncogene mRNA.
 60. The method of claim 14, wherein the mRNA encodes aviral protein.
 61. The method of claim 36, wherein the mRNA is acellular mRNA.
 62. The method of claim 36, wherein the mRNA is anoncogene mRNA.
 63. The method of claim 36, wherein the mRNA encodes aviral protein.